Publications since 1994

Calf thymus DNA adsorbed on rough gold substrate or atomically smooth gold (111) surface have been investigated by collecting their unique Raman fingerprints using either surface-enhanced Raman spectroscopy (SERS) or tip-enhanced Raman spectroscopy (TERS). Monolayer coverage of DNA strands adsorbed at both the irregular rough edges of evaporated gold grids and gold nanoparticles are detected by SERS. Much improved sensitivity that down to single DNA strand spectroscopic determination is accomplished by TERS, which provides an enhancement factor of at least 1400. With our experimental results, we propose that TERS is a promising technique for studying the DNA-drug molecule interaction on a level of single DNA strand.

Tip-enhanced near-field optical images and correlated topographic images of an organic semiconductor film (diindenoperylene, DIP) on Si have been recorded with high optical contrast and high spatial resolution (17 nm) using a parabolic mirror with a high numerical aperture for tip illumination and signal collection. The DIP molecular domain boundaries being one to four molecular layers (1.5 - 6 nm) high are resolved topographically by a shear-force scanning tip and optically by the simultaneously recording the 6 ·10 ⁵ times enhanced photoluminescence (PL). Excitation is 4 ·10 ⁴ times enhanced and the intrinsically weak PL-yield of DIP-film is 15 fold enhanced by the tip. The Raman spectra indicate an upright orientation of the DIP molecules. Enhanced PL contrast results from the local film morphology via stronger coupling between tip plasmon and exciton-polaritons in the DIP film.

A tightly focused radially polarized laser beam forms an unusual bimodal field distribution in an optical lambda/2-microresonator. We use a single-molecule dipole to probe the vector properties of this field distribution by tuning the resonator length with nanometer precision. Comparing calculated and experimental excitation patterns provides the three-dimensional orientation of the single-molecule dipole in the microresonator.

A method of combined thin-film deposition, electron beam lithography, and ion milling is presented for the fabrication of gold and silver nanostructures. The flexibility of lithographical processes for the variation of geometric parameters is combined with three-dimensional control over the surface evolution. Depending on the etching angle, different shapes ranging from cones over rods to cups can be achieved. These size- and shape-tunable structures present a toolbox for nano-optical investigations. As an example, optical properties of systematically varying structures are examined in a parabolic mirror confocal microscope.

We present a novel multiparameter microscopy approach allowing for both fluorescence and Raman imaging and spectroscopy of the same individual autofluorescent protein and its photoproduct by colocalization of the same species in the respective spectroscopic images. For the investigated bichromophoric autofluorescent protein DsRed_N42H we are able to assign different Raman spectra to the photoproducts of the distinct chromophores. Furthermore, a careful analysis of Raman spectra taken from native proteins in comparison to Raman spectra from photobleached species allows for a feasible estimation of the underlying photodegeneration processes of the individual spectral forms.

We present a general review of different microresonator structures and how they can be used in future device applications in modern analytical methods by tailoring the optical properties of single quantum emitters. The main emphasis is on the tunable ?/2-FabryPerot-type microresonator which we used to obtain the results presented in this article. By varying the mirror distance the local mode structure of the electromagnetic field is altered and thus the radiative coupling of fluorescent single quantum emitters embedded inside the resonator to that field is changed, too. As a result a modification of the optical properties of these quantum emitters can be observed. We present experimental as well as theoretical results illustrating this effect. Furthermore, the developed resonator can be used to determine the longitudinal position of embedded emitters with an accuracy of ?/60 by analyzing the excitation patterns of nano-sized fluorescent polymer spheres after excitation with a radially polarized doughnut mode laser beam. Finally, we will apply this resonator to a biological system and demonstrate the modification of Förster resonant energy transfer (FRET) efficiency by inhibiting the excited state energy transfer from the donor to the acceptor chromophore of a single DsRed protein.

A high-resolution near-field spectroscopic mapping technique is successfully applied to investigate the influence of thermal annealing on the morphology of a poly(3-hexylthiophene) and [6,6]-penyl-C61 butyric acid methyl ester (P3HT:PCBM) blend film. Based on the simultaneously recorded morphological and spectroscopic information, the interplay among the blend film morphology, the local P3HT:PCBM molecular distribution, and the P3HT photoluminescence (PL) quenching efficiency are systematically discussed. The PL and Raman signals of the electron donor (P3HT) and acceptor (PCBM) are probed at an optical resolution of approximately 10 nm, which allows the chemical nature of the different domains to be identified directly. In addition, the local PL quenching efficiency, which is related to the electron transfer from P3HT to PCBM, is quantitatively revealed. From these experimental results, it is proposed that high-resolution near-field spectroscopic imaging is capable of mapping the local chemical composition and photophysics of the P3HT:PCBM blends on a scale of a few nanometers.

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A versatile and efficient tip-enhanced spectroscopic imaging technique based on a parabolic mirror (PM) assisted near-field optical microscope is demonstrated. The replacement of the conventional objective lens with a parabolic mirror allows the non-restricted investigation of sample materials regarding their opacity. In addition, an improved signal collection efficiency and effective excitation of the longitudinal plasmonic oscillation in the tip apex are obtained. The capabilities of PM-assisted tip-enhanced Raman (TER) and photoluminescence (PL) imaging in distinguishing the individual domains made of different chemical components in poly (3-hexythiophene)/[6, 6]-penyl-C61 butyric acid methyl ester (P3HT/PCBM) solar cell blend film and in the investigation of the plasmonic properties of geometrically well-defined Au cones are demonstrated.

The poly(3-hexylthiophene) (P3HT) and [6,6]-phenyl C61 butyric acid methyl ester (PCBM) organic films are widely employed as electronic donor and acceptor in the field of organic film solar cell because of their high photovoltaic conversion efficiency. A home-built parabolic mirror assisted confocal and apertureless near-field optical microscope was used to investigate the degradation behavior of the film and to distinguish the donor and acceptor domains both topographically and optically. Under ambient condition, the degradation rates are decreased in the sequence of pristine P3HT, blend P3HT:PCBM film and pristine PCBM. N2 protection dramatically slows down the film degradation rate. Using confocal spectroscopic mapping, we are able to distinguish the local distributions of P3HT and PCBM. Micrometer PCBM aggregates were observed due to the thermal annealing process. Our experimental methods show the possibility to investigate morphology and the photochemistry properties of the organic solar cell films with high spatial resolution.

We present experimental results on changing the fluorescence spectrum of a single molecule by embedding it within a tunable optical microresonator with subwavelength spacing. The cavity length is reversibly changed across the entire visible range with nanometer precision by using a piezoelectric actuator. By varying its length, the local mode structure of the electromagnetic field is changed together with the radiative coupling of the emitting molecule to the field. Since mode structure and coupling are both frequency dependent, this leads to a renormalization of the emission spectrum of the molecule. Moreover, we use doughnut laser modes in the tunable microcavity to determine the longitudinal position of an isotropic emitter. By analyzing the excitation patterns resulting from the illumination of a single fluorescent bead in the focus of a radially polarized doughnut mode laser beam we can determine the longitudinal position of this bead in the microcavity with an accuracy of a few nanometers.

We present new results on single SiO2 nanoparticles (SiO2 NPs). NPs were obtained by full oxidation in water of silicon nanocrystals synthesized by CO2 laser pyrolysis of SiH4. Samples of SiO2 NPs embedded in low concentration in a thin polymer layer were prepared by spin-coating a dedicated solution on quartz cover slides. Using focused higher order laser modes, we determine the three-dimensional orientation of the nanoparticles transition dipole moment (TDM). The SiO2 NPs were found to possess a quite stable and randomly oriented TDM. However, characteristic dynamical effects featuring single NPs such as fluorescence intermittency and TDM flipping could also be observed. Photoluminescence (PL) spectroscopy of single SiO2 NPs revealed spectra with a double-peak structure consisting of a narrow zero-phonon line and a broader phonon band. The phonon band can be attributed to longitudinal optical phonons excited in the SiO2 network.

Silicon nanocrystals were synthesized by CO2 laser pyrolysis of SiH4. The fresh silicon nanopowder was oxidized in water to obtain SiO2 nanoparticles (NPs) exhibiting strong red-orange photoluminescence. Samples of SiO2 NPs embedded in low concentration in a thin polymer layer were prepared by spin-coating a dedicated solution on quartz cover slides. Using an argon ion laser at 488 nm with higher-order laser modes (azimuthally and radially polarized doughnut modes) for excitation, the three-dimensional orientation of the nanoparticles transition dipole moment was investigated in a confocal microscope. The linear transition dipole moment was found to be rather stable and randomly oriented. However, dynamical effects such as fluorescence intermittency and transition dipole moment flipping could also be observed. The spectral analysis of single SiO2 NPs revealed double-peak spectra consisting of a narrow zero-phonon line and a broader phonon band being associated with the excitation of longitudinal optical phonons in the SiO2 NP.

Using ambient atmosphere instead of pure nitrogen environment enabled efficient recording of room temperature fluorescence from single molecules of porphycenes, chromophores with a high triplet formation efficiency. Double hydrogen transfer between two chemically identical trans tautomers has been demonstrated for parent porphycene and three alkyl derivatives by the analysis of spatial patterns of the emission obtained after raster scanning the sample excited with an appropriately polarized laser beam. Because of tautomerization, fluorescence in porphycenes is due to two nearly orthogonal transition dipole moments. This property allows the spatial orientation of the single molecule chromophores to be determined using radially and azimuthally polarized laser beams as excitation sources.

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 sensors.
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.

Single molecule techniques have gained high interest over the past decade as they represent the ultimate limit of detection and sensitivity. Moreover, these studies benefit from the fact, that averaging over a huge number of species inherent in every ensemble study is overcome by single molecule methods and the specific response of a single emitter in its individual nano-environment can be addressed for example by fluorescent based methods. In addition, monitoring the temporal evolution and distribution of the fluorescence discovers spectral dynamics of the molecular emission. Extrinsic and intrinsic origins of the observed fluctuations can be discriminated and for example conformational changes of the molecular structure or the spectral influence of the flexibility of the molecular environment can be studied. Besides fluorescence based methods, surface enhanced Raman scattering has developed to be a powerful tool with single molecule sensitivity, providing direct insight into the molecular structure of a target system. Here, the careful analysis of the observed spectral fluctuations can provide information on the Raman enhancement process a molecule adjacent to a noble metal particle experiences.

Since the discovery of the technique in the early 1990s, single molecule spectroscopy has been used as a powerful tool to investigate and characterize fluorescent molecules, revealing insights into molecular behavior far beyond the information content that can be obtained by conventional ensemble studies. Several spectroscopic techniques have been established at the single molecule level, including spectrally resolved fluorescence, fluorescence lifetime investigations, or single molecule Raman measurements. However, the combination of two or more of these spectroscopies applied to the same individual molecule in multiparameter approaches yields a deeper understanding of molecular systems. In this contribution, we present our results of combined spectrally- and time-resolved fluorescence microscopy of the intrinsic fluorescence energy transfer (FRET) system of the red fluorescent protein DsRed. Correlating the results obtained from the two spectroscopic techniques, we are able to determine all relevant parameters to describe the energy transfer processes within the DsRed system without any further assumptions. We further discuss fluorescence and surface enhanced Raman scattering (SERS) spectroscopy of the same individual DsRed unit, which can help to propose mechanisms for photodegeneration of the distinct chromophores involved.

We investigate experimentally the modifications of the fluorescence properties of the bichromophoric fluorescent resonance energy transfer (FRET) system DsRed imposed by optical confinement. The confinement-condition is realized by a novel /2-microresonator that modifies the local photonic mode density in the vicinity of the proteins while maintaining a physiological environment for the embedded biological molecules. The experimental ratio of the fluorescence intensities and lifetimes, respectively, of donor and acceptor chromophores varies by up to a one order of magnitude as we vary the mirror spacing of the microresonator with nanometer-precision. Since these ratios determine the FRET efficiency, we modify the yield of the excited state energy transfer in rigidly coupled FRET pairs without chemically or physically perturbating the chromophoric subunits. We show that the microresonator-controlled inhibition of the acceptor fluorescence results in a loss of transfer efficiency of excited state energy from donor to acceptor, an effect that enables the spectral isolation and efficient observation of donor chromophores both in DsRed ensembles and on the single protein level. This constitutes an important application of microcavity-enhanced single molecule spectroscopy of biological systems and shows the potential of optical confinement for applications in nano-biophotonics.

We analyzed the scattering patterns of individual Au nanorods detected by means of confocal interference scattering microscopy in combination with a higher order laser mode. We placed the Au nanorods at the interface between two dielectric media and examined the influence of different interfaces on the signal amplitude, the signal-to-noise ratio as well as on the precision of topological measurements. Approaching the index matching regime allows for topological measurements with high accuracy minimizing the acquisition time. We were also able to track the position and the orientation of particles embedded in water even when they were not thoroughly sticking to the glass surface. These results underscore the potential of the presented technique for applications in life sciences.

We present experimental and theoretical results on changing the fluorescence emission spectrum of a single molecule by embedding it within a tunable planar microcavity with subwavelength spacing. The cavity length is changed with nanometer precision by using a piezoelectric actuator. By varying its length, the local mode structure of the electromagnetic field is changed together with the radiative coupling of the emitting molecule to the field. Because mode structure and coupling are both frequency dependent, this leads to a renormalization of the emission spectrum of the molecule. We develop a theoretical model for these spectral changes and find excellent agreement between theoretical prediction and experimental results.

The exact localization of a quantum emitter in a transparent dielectric medium is an important task in applications of precision confocal microscopy. Therefore we use a planar metallic subwavelength microcavity which can be reversibly tuned across the whole visible range with the transparent medium between the cavity mirrors. By analyzing the excitation patterns resulting from the illumination of a single fluorescent bead with a radially polarized doughnut mode laser beam we can determine the longitudinal position of this bead in the microcavity with an accuracy of a few nanometers.

We investigate experimentally and theoretically the fluorescence emitted by molecular ensembles as well as spatially isolated, single molecules of an organic dye immobilized in a quasi-planar optical microresonator at room temperature. The optically excited dipole emitters couple simultaneously to on-and off-axis cavity resonances of the microresonator. The multi-spectral radiative contributions are strongly modified with respect to free (non-confined) space due to enhancement and inhibition of the molecular spontaneous emission (SpE) rate. By varying the mirror spacing of the microresonator on the nanometer-scale, the SpE rate of the cavity-confined molecules and, consequently, the spectral line width of the microresonator-controlled broadband fluorescence can be tuned by up to one order of magnitude. Stepwise reducing the optical confinement, we observe that the microresonator-controlled molecular fluorescence line shape converges towards the measured fluorescence line shape in free space. Our results are important for research on and application of broadband emitters in nano-optics and -photonics as well as microcavity-enhanced (single molecule) spectroscopy.

A sharp-tipped gold nanocone and the vertically aligned metallic tip of a near-field optical microscope together form a three-dimensional optical antenna with a highly controllable gap. Confocal measurements with different laser modes show the efficient axial excitation of the cones with a longitudinally polarized field. In the antenna configuration, extremely strong field enhancement up to a factor of 100 is obtained by tuning the gap between the two sharp tips down to few nanometers.

We study spatially isolated, individual gold nanorods placed at a planar interface between two dielectric media using confocal interference scattering microscopy in combination with higher order laser modes. Approaching refractive index matching conditions, we observe that the elastic scattering patterns of individual nanorods exhibit an exponential increase of both the scattering intensity and the signal-to-background ratio. In case refractive index matching conditions are fullfilled, the data acquisition rates are maximized and suitable for in-vivo biological measurements. In all cases, the characteristic two-lobe shape of the scattering patterns of single nanorods remains unchanged while the sign of the image contrast is a direct consequence of the refractive index variation occurring at the interface.

The red fluorescent protein from DsRed from Discosoma reef coral exhibits complex photophysics. One key reason for this is, that DsRed forms obligate tetrameric units containing green and red emitting monomers in random composition. Experimental investigations have proven that these ifferent chromophores within one tetramer are coupled by fluorescence resonance energy transfer (FRET) and that the observed strong red emission is due to a non-radiative energy transfer from the green to the red chromophore when the green chromophore is exclusively excited. Ensemble studies can only provide averaged data on statistical mixtures of tetramers with different compositions, since it is impossible to separate the tetramers into functional monomers containing only red or green emitting chromophores. We present here the results of DsRed multiparameter single molecule spectroscopy. By combining spectral and time domain spectroscopy we were able to isolate single tetramers containing only green chromophores and thus record the fluorescence lifetime of the green emitting species without interference form FRET to the red chromophore for the first time. The fluorescence lifetime for the green chromophore of DsRed is remarkably longer than for the green fluorescent protein, GFP, which is a chemical analogue to the green chromophore in DsRed. Based on our single protein experiments we can derive a complete set of spectroscopic parameters to describe Förster energy transfer in the DsRed system without any further assumptions. Hence in combination with X-ray studies our data allow for an accurate quantitative description of the radiative and non-radiative relaxation processes in DsRed proteins.

We evaluate the field distribution in the focal spot of the fundamental Gaussian beam as well as radially and azimuthally polarized doughnut beams focused inside a planar metallic sub-wavelength microcavity using a high numerical aperture objective lens. We show that focusing in the cavity results in a much tighter focal spot in longitudinal direction compared to free space and in spatial discrimination between longitudinal and in-plane field components. In order to verify the modeling results we experimentally monitor excitation patterns of fluorescence beads inside the lambda/2-cavity and find them in full agreement to the modeling predictions. We discuss the implications of the results for cavity assisted single molecular spectroscopy and intra-cavity single molecular imaging.

We have investigated the radiation patterns formed by a quasi-planar optical lambda/2-microresonator enclosing fluorescent dye molecules that were immobilized in a polymer film between two silver mirrors. Using time-resolved widefield imaging microscopy, we observed laterally confined transversal modes that occurred in the optically pumped microresonator area, exhibiting strong intensity fluctuations. The measured diameter of the isolated spatial modes was found to be 0.5 mu m in agreement with theoretical predictions. The instability of the spatial mode emission patterns originates from the triplet-state-induced fluorescence intensity fluctuations of cavity-coupled collective molecular excited states.

We demonstrate experimentally and theoretically that a parabolic mirror (PM) with a high numerical aperture (NA) of 1 focuses a radially polarized laser mode to the smallest diffraction-limited spot at a fixed NA and wavelength, having an area of 0.134 lambda(2). The measurements were performed with a confocal microscope, using the PM as a focusing and collecting element. The results stand in accordance with the theoretical calculations presented by Davidson and Bokor [Opt. Lett. 29, 1318 (2004)], who predicted a reduction in the total focal spot size of 43% as compared with an aplanatic lens.

We have investigated the influence of the plasmon resonances of individual, spatially isolated gold nanoparticle aggregates on their emission properties using combined optical confocal and dark field scattering microscopy and spectroscopy. The emission intensity of the same aggregate is enhanced by up to 1 order of magnitude if the emission wavelengths overlap with the plasmon resonance in the corresponding white light scattering spectrum. Regardless of the specific geometry of an individual aggregate, the in situ measurement of the plasmon characteristics delivers unique information about its potential as a substrate for surface-enhanced spectroscopy and allows its characterization as a nanoscatterer, nanoemitter, or local heater.

Detecting efficiently the plasmon-enhanced Raman signal of molecules created in the nanometre-sized gap between a metal nanoparticle or the apex of a sharp tip and a metal surface is the key problem in particle- or tip-enhanced local surface spectroscopy (Pettinger et al., 2004; Roth et al., 2006). The optical excitation field has to be polarized along the gap, and the field emerging from the gap has to be observed from the side. These geometrical restrictions usually limit the numerical aperture of the lens used for exciting the gap and collecting the scattered photons created in the gap. We present a novel method to overcome this problem. The solution is based on a confocal optical microscope with a high numerical aperture parabolic mirror for excitation and detection. Localized plasmons can be efficiently excited parallel to the surface normal by illuminating the parabolic mirror with a radially polarized doughnut mode and the field emerging sidewise from the gap can be efficiently collected by the rim of the parabolic mirror and directed to the detection system. First results on particle- and tip-enhanced Raman spectroscopic measurements of benzotriazole molecules adsorbed on gold films are presented.

We demonstrate a novel optical method for characterizing single Au nanoparticles by acquiring their scattering patterns. This technique combines confocal microscopy and higher-order laser modes for detecting the light scattered by sub-wavelengthsized nanoobjects. The optical patterns are generated by the coherent superposition of the field scattered by individual metallic particles and the excitation field reflected at the cover slideair interface and provide information about the particles' position, orientation, size and shape. Detectable changes in the full width at half maximum (FWHM) of the signal intensity permit to distinguish between 20- and 60-nm diameter Au spheres. The confocal images are also very sensitive to the particle's geometry and polarizability, that is, Au nanospheres, Au nanorods and triangular Au nanoplates give different characteristic patterns if the excitation wavelength is varied.

A novel near-field optical microscope based on a parabolic mirror is used for recording high-resolution tip-enhanced photoluminescence (PL) and Raman images with unprecedented sensitivity and contrast. The measurements reveal small islands on the Au surface with dimensions of only a few nanometres with locally enhanced Au PL. These islands appear as nanometre-sized hot spots in tip-enhanced Raman microscopy when benzotriazole molecules adsorbed on the Au surface serve as local sensors for the optical field. The spectra show that localized plasmons are the cause of both the locally enhanced Au PL and enhanced Raman scattering. This finding suggests that the dispersive background in the surface-enhanced Raman spectra can be explained simply by the enhanced Au PL in the gap. Furthermore, our results show that the surface flatness must be better than 1 nm, to provide an optically homogeneous substrate for near-field enhanced PL and Raman spectroscopy.

We use spectrally-resolved room temperature single molecule spectroscopy to yield insights into the occurrence and dynamics of spectral forms of single tetramers of DsRed and its variants DsRed2, Fluorescent Timer, DsRed_N42H and AG4. The red-emitting chromophore in DsRed and all studied variants readily converts into a high quantum efficiency super-red emitting form. We propose the existence of two super-red forms of different quantum efficiencies. The observed emission from the green-emitting chromophore is consistent with bulk spectroscopy. We further observe distinct new spectral forms from each variant, which we attribute to a photoinduced chemical reaction leading to a truncated form of the red-emitting chromophore analogous to the chromophore in the visible fluorescent protein zFP538. Our results have implications for the accurate interpretation of biological and biochemical processes illuminated by fluorescent proteins as well as for choosing appropriate experimental configurations.

We present a novel scattering microscopy method to detect the orientation of individual silver nanorods and to measure their relative distances. Using confocal microscopy in combination with either the fundamental or higher order laser modes, scattering images of silver nanorods were recorded. The distance between two individual nanorods was measured with an accuracy in the order of 1 nm. We detected the orientation of isolated silver nanorods with a precision of 0.5 degree that corresponds to a rotational arch of about 1 nm. The results demonstrate the potential of the technique for the visualization of non-bleaching labels in biosciences.

We studied spatially isolated single-walled carbon nanotubes (SWNTs) immobilized in a quasi-planar optical lambda/2-microresonator using confocal microscopy and spectroscopy. The modified photonic mode density within the resonator is used to selectively enhance or inhibit different Raman transitions of SWNTs. Experimental spectra are presented that exhibit single Raman bands only. Calculations of the relative change in the Raman scattering cross sections underline the potential of our microresonator for the optical control of nonequilibrium phonon populations in SWNT.

The authors have investigated the conformational structure of the ferroelectric liquid crystal compound 4-3-methyl- 2-chloropentanoyloxy- 4(')-hexyloxy- biphenyl also known under the abbreviations 3M2CPHOB and C6 using vibrational (IR and Raman) spectroscopy. The measured spectra exhibit two bands corresponding to the C = O stretching vibration that are separated by 20 cm(-1). In contrast, the molecular structure comprises only one such group. They assigned the two bands to different conformers that coexist in a temperature range between 25 and 65 degrees C covering the entire mesophase of this material. This assignment is strongly confirmed by calculated vibrational spectra based on the density functional theory.

We use two-beam interferometry in combination with confocal microscopy for Roman and fluorescence studies of spatially isolated single-walled carbon nanotubes and single dye molecules. We investigate the potential of optical Fourier transform spectroscopy for the spectral characterization of single molecules and molecular nanostructures. We demonstrate that it is possible to obtain the temporal coherence characteristics as well as reliable spectroscopic data of the single photon fluorescence emission of an isolated molecule from one measured interferogram, even though the molecule exhibits intensity fluctuations and spectral jumps.

The authors studied spatially isolated terrylene molecules immobilized in a quasiplanar optical lambda/2-microresonator using confocal microscopy and spectroscopy at variable temperatures. At T=1.8 K, they observed individual molecules relaxing into microresonator-allowed vibronic levels of their electronic ground state by emission of single fluorescence photons. Coupling the purely electronic transition of embedded molecules to the longitudinal photonic mode of the microresonator resulted in an ultimate spectral narrowing and an increased collection efficiency of the emitted single photon wave trains. (c) 2007 American Institute of Physics.

Optical spectroscopy provides a wealth of information on the electronic and vibronic states of biological materials and surfaces, thus allowing for a detailed analysis of the sample constituents. Furthermore, information on photoprocesses such as excited-state relaxation, charge transfer and coupling of individual quantum systems, as for example, in light-harvesting complexes, can also be obtained. Advances in near-field optics open up new means to overcome the diffraction limit and extend the range of optical measurements to the length scales of most nanosystems. In this paper, we describe a near-field microscopic technique that relies on the enhanced electric field near a sharp, laser-irradiated metal tip. This confined light source can be used for highly localized excitation of photoluminescence (PL) and Raman scattering. We study the properties of the enhanced fields and demonstrate PL and Raman imaging of single-walled carbon nanotubes (SWCNTs) with sub-15 mn resolution. The existing studies on biomaterials using tip-enhanced techniques in the literature are reviewed and potential applications together with the requirements on sample materials are discussed.

Near-field photoluminescence spectroscopy was used to study the electronic properties of semiconducting Single-Walled Carbon Nanotubes in different environments. A sharp laser-illuminated metal tip was raster scanned over the sample and served as a strongly confined excitation source. We observed localization of photoluminescence and variations of emission energies along nanotubes on a length scale of about 30 nm.

Homoleptic and heteroleptic ruthenium trisphenanthrolines 1b-d were prepared with azacrown ethers attached to the 4,7-positions of the phenanthrolines to maximise the electronic communication between the ruthenium and the crown ethers as complexation sites. Redox and spectral data were processed to explain the non-steady trends in the absorption and emission spectra in the series 1b-d. Addition of Ba2+ entailed large shifts in the redox potential (up to 370 mV) and in the emission spectra (up to 87 nm). Due to the crowded situation of the azacrown ether units in 1d, this complex showed a non-linear behaviour both in the redox and emission properties upon loading with Ba2+ that is postulated to originate from the intermediate formation of sandwich type complexes.

We present a new method for the imaging of single metallic nanoparticles that provides information about their shape and orientation. Using confocal microscopy in combination with higher order laser modes, scattering images of individual particles are recorded. Gold nanospheres and nonorods render characteristic patterns reflecting the different particle geometries. In the case of nanorods, the scattering patterns also reveal the orientation of the particles. This novel technique provides a promising tool for the visualization of nonbleaching labels in the biosciences.

When analyzing the emission of a large number of individual chromophores embedded in a matrix, the spread of the observed parameters is a characteristic property for the particular chromophore-matrix system. To quantitatively assess the influence of the matrix on the single molecule emission parameters, it is imperative to have a system with a well-defined chromophore nanoenvironment and the possibility to alter these surroundings in a precisely controlled way. Such a system is available in the form of the visible fluorescent proteins, where the chromophore nanoenvironment is defined by the specific protein sequence. We analyze the influence of the chromophore embedding within this defined protein environment on the distribution of the emission maximum wavelength for a number of variants of the fluorescent protein DsRed, and show that this parameter is characteristic of the chromophore-protein matrix combination and largely independent of experimental conditions. We observe that the chemical changes in the vicinity of the chromophore of different variants do not account for the different distributions of emission maximum positions but that the flexibility of the chromophore surrounding has a dominant role in determining the distribution. We find, surprisingly, that the more rigid the chromophore surrounding, the broader the distribution of observed maximum positions. We hypothesize that, after a thermally induced reorientation in the chromophore surrounding, a more flexible system can easily return to its energetic minimum position by fast reorientation, while in more rigid systems the return to the energetic minimum occurs in a stepwise fashion, leading to the broader distribution observed.

We present a new microcavity design which allows for efficient detection of single molecules by measuring the molecular fluorescence emission coupled into a resonant cavity mode. The Fabry-Perot-type microresonator consists of two silver mirrors separated by a thin polymer film doped with dye molecules in ultralow concenctration. By slightly tilting one of the mirrors different cavity lengths can be selected within the same sample. Locally, on a pm scale, the microcavity still acts as a. planar Fabry-Perot resonator. Using scanning confocal fluorescence microscopy, single emitters on resonance with a single mode of the microresonator can be spatially addressed. Our microcavity is demonstrated to be well-suited for investigating the coupling mechanism between single quantum emitters and single modes of the electromagnetic field. The microcavity layout could be integrated in a lab-on-a-microchip design for ultrasensitive microfluidic analytics and can be considered as an important improvement for single photon sources based on single molecules operating at room temperature.

In this contribution, we apply a microscopic technique that relies on the enhanced electric field near a sharp, laser-irradiated metal tip that acts as a highly confined light source. The tip is used for the local excitation of the optical response of single-walled carbon nanotubes (SWNT) deposited on glass. We demonstrate photoluminescence and Raman imaging of the same SWNTs with a spatial resolution of about 10 nm.

The interest in single molecule spectroscopy by means of surface enhanced Raman scattering (SERS) has grown enormously in the last five years. Unfortunately, the basic mechanisms leading to the giant enhancement of the Raman signals are still not understood. Almost every group working in the field of single molecule SERS uses silver or gold colloids prepared by a chemical reduction method, to which a highly diluted solution of the target molecules is added. After that, the colloids, are deposited gravitationally onto a glass cover slide. This leads to clusters of several nanoparticles with a large distribution regarding size and shape as well as the Raman efficiency, and the systematic investigation of sm-SERS suffers from this. Our aim was to develop a method to deposit silver clusters showing a high enhancement factor in a controlled way. For this purpose we used an electrochemical double-pulse method, which allows to control the particle size and density by systematic variation of the pulse parameters. We also compared measurements obtained with these surfaces to measurements made with colloids at the single molecule level.

We present simultaneous near-field photoluminescence (PL) and Raman imaging of single-walled carbon nanotubes (SWNTs) with a spatial resolution better than 15 nm. Highly localized excitation is used to visualize the spatial extent of the contributing excited states. For SWNTs on glass, we typically observe highly confined PL from short segments of about 20 nm in length. The PL from micelle-encapsulated SWNTs on mica is extended along the tube up to several hundreds of nanometers. We find that near-field enhancement is much stronger for photoluminescence than for Raman scattering, an observation that is explained by the low intrinsic quantum yield of SWNTs.

Abstract

Surface-enhanced resonance Raman scattering (SERRS) spectra of various rhodamine dyes, of pyronine G and thiopyronine adsorbed on isolated silver clusters were recorded at the ensemble level and at the single-molecule level with a high-resolution confocal laser microscope equipped with a spectrograph and a CCD-detector. Comparing single-molecule spectra with ensemble spectra, various inhomogeneous spectral features, such as line splitting, spectral wandering, spectral diffusion and abrupt spectral jumps between different metastable spectral states, are revealed in temporal sequences of spectra; these affect both the frequency positions and the relative intensities of the vibronic bands. Resonance enhancement is investigated with respect to single-molecule surface-enhanced Raman scattering (SERS) spectroscopy and is found to be responsible for approximately three. orders of magnitude in sensitivity. A significant influence of the substituents on the single-molecule SERRS sensitivity is found, showing that various chemical effects are responsible for surface enhancement in addition to the electromagnetic enhancement effect.

We present for the first time cavity-controlled fluorescence spectra and decay curves of single dipole emitters interacting at room temperature with the first longitudinal mode of a Fabry-Perot microcavity offering a l/2-spacing between its silver mirrors. The spontaneous emission rate of individual dye molecules was found to be enhanced by the Purcell effect by up to three times compared to the rate in free space in agreement with theoretical predictions. Moreover, our new microcavity design was found to provide long-term stability and single-molecule sensitivity under ambient conditions for several months without noticeable reduction of the cavity Q-value. We consider this as a significant advance for single-photon sources operating at room temperature.

The dynamics of excitons in individual semiconducting single-walled carbon nanotubes (SWNTs) were studied using time-resolved photoluminescence (PL) spectroscopy. The PL decay from tubes of the same (n,m) type was found to be mono-exponential, however, with lifetimes varying between less than 20 ps and 200 ps from tube to tube. Competition of non-radiative decay of excitons is facilitated by a thermally activated process, most likely a transition to a to a low-lying optically inactive trap state that is promoted by a low-frequency phonon mode.

Tautomerization is a fundamental process in chemistry and biology, where it plays a major role in vision and enzymatic reactions. Usually, extensive spectroscopic ensemble studies are required to identify a tautomeric equilibrium. For instance, indirect evidence for the fast motion of the two inner hydrogen atoms between the nitrogen atoms has been deduced for porphycene, a constitutional isomer of porphyrin, from complex NMR1, and fluorescence spectroscopy studies in a solid host for both ground and excited singlet states.

Novel insights into the electronic structure of carbon nanotubes are obtained using single molecule fluorescence spectroscopy. Fluorescence spectra from single nanotubes are well described by a single, Lorentzian lineshape. Nanotubes with identical structures fluoresce with different energies due to local electronic perturbations. Carbon nanotube fluorescence unexpectedly does not show any intensity or spectral fluctuations at 300K. The lack of intensity blinking or bleaching demonstrates that carbon nanotubes have the potential to provide a stable, single molecule infrared photon source, allowing for the exciting possibility of applications in quantum optics.

It is known from ensemble spectroscopy at cryogenic temperatures that variants of the Aequorea green fluorescent protein (GFP) occur in interconvertible spectroscopically distinct forms which are obscured in ensemble room temperature spectroscopy. By analyzing the fluorescence of the GFP variants EYFP and EGFP by spectrally resolved single-molecule spectroscopy we were able to observe spectroscopically different forms of the proteins and to dynamically monitor transitions between these forms at room temperature. In addition to the predominant EYFP B-form we have observed the blue-shifted I-form thus far only seen at cryogenic temperatures and have followed transitions between these forms. Further we have identified for EYFP and for EGFP three more, so far unknown, forms with red-shifted fluorescence. Transitions between the predominant forms and the red-shifted forms show a dark time which indicates the existence of a non fluorescent intermediate. The spectral position of the newly-identified red-shifted forms and their formation via a non fluorescent intermediate hint that these states may account for the possible photoactivation observed in bulk experiments. The comparison of the single-protein spectra of the red-shifted EYFP and EGFP forms with single-molecule fluorescence spectra of DsRed suggest that these new forms possibly originate from an extended chromophoric pi-system analogous to the DsRed chromophore.

Various switching processes with jumps between two or more spectral states were observed for single molecules of the fluorescent dyes PI and DAPI in polystyrene at room temperature. Switching processes were found in the temporal trajectories of almost each spectral parameter. Statistical analyses on the distribution of jumpwidths and on correlations between them were performed. By this means a discrimination between extrinsic and intrinsic mechanisms, which trigger the observed switching dynamic, has been possible in some cases even without anticipated assumption on the nature of the mechanism itself. It has been found that PI and DAPI behave considerably different in single molecule spectroscopy, opposed to their almost identical appearance on bulk level.

Surface enhanced resonance Raman spectra of various rhodamine dyes, of pyronine G and thiopyronine adsorbed on isolated silver clusters were recorded at the single-molecule level with a high-resolution confocal laser microscope equipped with a spectrograph and a CCD-detector. The comparison of the spectra shows that the xanthene chromophore dominates the surface enhanced resonance Raman scattering spectra and wavelength selective excitation shows that resonance excitation contributes significantly to the spectral enhancement.

We use near-field Raman imaging and spectroscopy to study localized vibrational modes along individual, single-walled carbon nanotubes (SWNTs) with a spatial resolution of 10-20 nm. Our approach relies on the enhanced field near a laser-irradiated gold tip which acts as the Raman excitation source. We find that for arc-discharge SWNTs, both the radial breathing mode (RBM) and intermediate frequency mode (IFM) are highly localized. We attribute such localization to local changes in the tube structure ( n, m). In comparison, we observe no such localization of the Raman active modes in SWNTs grown by chemical vapor deposition (CVD). The direct comparison between arc-discharge and CVD-grown tubes allows us to rule out any artifacts induced by the supporting substrate.

Near-field Raman spectroscopy with high spatial resolution is applied to study singlewalled carbon nanotubes (SWNT) on glass. A sharp, laser illuminated metal tip acts as near-field source causing an enhanced Raman signal within close proximity of the tip. We present optical images of different Raman modes with a resolution below 20 nm. Using tip-enhanced Raman spectroscopy, different tubes structures are distinguished on the nanoscale and highly localized phonon modes are revealed.

We present near-field Raman spectroscopy and imaging of single isolated single-walled carbonanotubes. The surface modification of single-crystalline silicon induced by single 130 femtosecond (fs) Ti:sapphire laser pulses (wavelength 800 nm) in air is investigated by means of micro Raman spectroscopy (mu-RS), atomic force microscopy and scanning laser microscopy. Depending on the laser fluence, in some regions the studies indicate a thin amorphous top-layer as well as ablated and recrystallized zones. The single-pulse threshold fluences for melting, ablation and polycrystalline recrystallization are determined quantitatively. Several different topographical surface structures (rims and protrusions) are found. Their formation is discussed in the context of recent studies of the laser irradiation of silicon. In combination with a thin-film optical model, the thickness of the amorphous layer is determined by two independent and nondestructive optical methods to be in the order of several 10 nm. (C) 2003 Elsevier B.V. All rights reserved.

Near-field Raman spectroscopy with high spatial resolution is used to study single-molecules. For laser spectroscopy at variable temperatures with high spatial resolution a combined scanning near-field optical and confocal microscope was developed. Rhodamine 6G (R6G) dye molecules dispersed on silver nano-particles or nano-clusters were investigated. For optical excitation of the molecules, either an aperture probe or a focused laser spot in confocal arrangement were employed. Raman spectra in the wavenumber range between 300 cm(-1) and 3000 cm(-1) at room temperatures down to 8.5 K were recorded. Many of the observed Raman lines can be associated with the structure of the adsorbed molecule. Intensity fluctuations in spectral sequences were observed down to 77 K and are indicative of single molecule sensitivity.

Parabolic mirrors with a high numerical aperture can be conveniently used to produce highly confined optical fields in the focal region. Furthermore, these fields can have interesting polarization behaviour due to the high numerical aperture. In particular, if the mirror is illuminated with a size matched radially polarized or azimuthally polarized doughnut mode, the electric field has in the focal region almost exclusively a longitudinal or a transverse polarization component. Such field distributions are interesting for applications in confocal or near-field optical microscopy. Here we present experimental results where we have probed some of these field distributions by raster scanning a fine gold tip in nanometer steps through the focal region and detecting the scattered light intensity. The measured intensity patterns are compared with corresponding vector-field calculations.

We studied the emission of mutants of the red fluorescent protein DsRed by room temperature single molecule fluorescence spectroscopy. Bulk samples of the DsRed variant E8 show mixed green and red fluorescence of equivalent intensities individually spectrally similar to arrested green and mature red fluorescent forms of DsRed. Investigations at the single molecule level indicate that, like DsRed, E8 is not monomeric at single molecule concentrations. The entities visualized are composed of green and red emitting proteins without a fixed ratio of green to red fluorescing units. We find indications for only weak, if any, fluorescence resonance energy transfer (FRET) between red and green chromophores within one E8 entity.

Surface-enhanced resonance Raman scattering (SERRS) of rhodamine 6G (R6G) adsorbed on isolated colloidal silver clusters has been studied down to the single-molecule level with a high-resolution confocal optical microscope equipped with a spectrometer and a cooled CCD-camera. At the single-molecule level the SERRS-spectra recorded as a function of time reveal inhomogeneous behaviour such as on/off blinking, spectral diffusion and intensity fluctuations of vibrational lines, and even splitting of some lines within the spectrum of one molecule.

A novel high-resolution stage scanning confocal microscope for fluorescence microscopy and spatially resolved spectroscopy with a high numerical aperture (NA ¯ 1) parabolic mirror objective is investigated. A spatial resolution close to the diffraction limit is achieved. As microscopic fluorescent test objects, dye-loaded zeolite microcrystals (diameter approx. 0.4 æm) and single fluorescent molecules were used. Confocal fluorescence images show a spatial resolution of Dx = 0.8l both at room temperature and at 1.8 K. Imaging of a quasi-point light source and focusing by the parabolic mirror were investigated experimentally and theoretically. Deviations be-tween the theoretical results for a perfect parabolic mirror and the experi-mental results can be attributed to small deviations of the mirror profile from an ideal parabola.

The single-molecule fluorescence properties of the two perylenediimide dyes, 9-amino-N-(2,6diisopropylphenyl)perylene-3,4-dicarboximide (API), and the derivative, N,N-di(tert-butoxycarbonyl)-API (DAPI) are investigated in the amorphous host polystyrene at room temperature. For API three spectroscopically different types of spectra can be distinguished, two of them can be assigned to the off-resonance and the in-resonance conformer of the amino group and the chromophoreïs p-electron system, repectively. The bathochromic third spectrum is suggested to originate also from an off-resonance conformer. In accordance with the sterically demanding substituents only off-resonance spectra are found for DAPI. Not being limited by the inhomogeneous spectral distribution, many molecules show clearly resolved vibronic structure and can be characterized by their spectral means, the vibronic maxima positions, and the vibronic band gaps. Sequences of consecutive spectra reveal different forms of dynamic behaviour such as diffusion and abrupt jumps both, in the spectral domain and in the intensity trajectories. These can be summarized in a rough classification of the observed dynamic phenomena.

We explore the diffraction limited focusing and confocal imaging properties of a high NA parabolic mirror for confocal imaging and spectroscopy of nanoparticles and single molecules. Vector field calculations of the electric fields near focus for both linear and radially polarized illumination are discussed and show that the optical field can be similar tightly focused as in the case of a high NA objective lens. Furthermore they show that a high NA parabolic mirror allows an easy orientation of the polarization of the illuminating light in all spatial directions. The simulation of confocal imaging of single molecules is discussed and yields, that the use of radially polarized excitation light gives an easy access to their orientations.

Abstract

The orientation of the S-1 <-- S-0 ,pi* transition dipole moments of oxonine (Ox(+)), pyronine (Py+), and POPOP (5,5'-diphenyl-2,2'-p-phenylenebis(oxazole)) in the channels of zeolite L crystals was investigated by means of fluorescence microscopy and single-crystal imaging. Qualitative observations led to the result that the transition moment of POPOP is aligned along the c-axis of the hexagonal crystals whereas the fluorescence of Ox(+) and Py+ is not. More detailed investigations on Ox(+) showed a cone-shaped distribution of the transition moments with a half-cone angle of 72 degrees. The orientation of the transition dipole moment for all of these molecules is parallel to the molecules' long axis. We found by means of space-filling arguments that POPOP, the van der Waals length of which is about 21 Angstrom, can only be aligned along the channel axis. This is in full agreement with the observed fluorescence anisotropy. For Ox(+) and Py+, geometrical arguments based on the zeolite L structure give room for only two possible arrangements of the molecules' long axis: a half cone angle of up to 40 degrees for Ox(+) and up to 30 degrees for Py+, and an angle of about 90 degrees for both of them with respect to the c-axis of zeolite L. The surprising discrepancy between geometrical considerations and the results of the fluorescence measurements can be explained by assuming that Ox(+) and Py+ are exposed to a considerable anisotropic electrical field in the zeolite channels.

Single molecule spectral dynamics originate from changes (1) within the chromophore and (2) in the chromophoric environment, hence are intrinsic and extrinsic. To interpret spectral phenomena of intrinsic origin, assignment of fluorescence behaviour to distinct molecular states is required. We realized this by means of a perylene based chromophore with an amino substituent. The fluorescence spectrum of this dye depends strongly on the amino group conformation relative to the chromophore. Thus different conformers of the chromophore can be distinguished. Here we evidence the occurrence of spectrally defined conformers and report transitions between them, revealed by single molecule spectroscopy.

Sequences of single-molecule fluorescence spectra are presented with integration times per spectrum as short as 25 ms. By reducing averaging effects in the time domain, the vibronic bands appear better resolved, often they show distinct bands instead of a shoulder in time-averaged spectra. Furthermore, we note that the fluctuation of the intensity ratio of the vibronic bands can considerably contribute to room-temperature spectral diffusion. This is in contrast to spectral diffusion of narrow homogenous Lorentzian absorption line profiles at cryogenic temperatures.

Abstract

The fluorescence emission of single rhodamine dye molecules (rhodamine 6G and rhodamine 630) at room temperature was analyzed by using scanning confocal laser microscopy in conjunction with polarization analysis, fluorescence spectroscopy, time-resolved detection (minutes to microseconds), and excitation saturation. Results are presented and discussed 1) for samples with dye molecules at the glass-air interface and 2) covered with an additional thin protective polymer film (polyvinylbutyral). Under the polymer layer, the single-molecule fluorescence was more stable than the glass-air interface. This result may be explained by fewer spontaneous variations of the fluorescence rate, polarization changes, spectral shifts, and longer photochemical lifetimes.

Scanning a point light source in close proximity over a sample and recording the scattered or transmitted light intensity point by point allows one to record optical images with a resolution not limited by diffraction, An overview of this technique called scanning near-field optical microscopy (SNOM or NSOM) is given with emphasis on cell- and microbiology. After an introduction, where the basic features of the technique are explained, illustrative examples are presented, such as a HeLa cell, fluorescence labelled human chromosomes, super resolution fluorescence imaging, single molecule imaging and fluorescence resonance energy transfer between a single pair of dye molecules.

Scanning near-field optical microscopy (SNOM) is an optical microscopy whose resolution is not bound to the diffraction limit. It provides chemical information based upon spectral, polarization and/or fluorescence contrast images. Details as small as 20 nm can be recognized. Photophysical and photochemical effects can be studied with SNOM on a similar scale. This article reviews a good deal of the experimental and theoretical work on SNOM in Switzerland.

We report on the imaging of single dye molecules in thin polymer films under ambient conditions by means of scanning near-field optical microscopy. The long-term mechanical stability and high detection efficiency of our instrument allow the imaging of single dye molecules over several hours with a high signal-to-noise ratio. The local excitation by the near-field tip, the low excitation power and the fact that the dye molecules are embedded in the three dimensional polymer structure drastically reduce photodestruction as compared with conventional microscopy. The observation of many molecules in the same film allows to probe them under identical experimental conditions and to statistically analyze their global behavior. We find that the molecules diffuse and rotate within the polymer matrix. The single dye molecules act as nanometer-sized probes and reflect the local structure of the polymer.

The time evolution of the microscopic lateral diffusion and dynamic reorientation of individual dye molecules dispersed in a thin polymer film is probed by means of scanning near-field optical microscopy at room temperature and ambient conditions, On a sub-micrometer scale we identify regions where the diffusion is drastically non-random, while the statistical average of the trajectories of 97 molecules in a 4 mu m x 4 mu m section displays random-walk behavior.

We have measured the temperature profile of aluminum coated fiber tips used for illumination-mode scanning near-field optical microscopy as a function of the optical input power with a micron sized thermocouple. The temperature coefficients vary from 20 K/mW for tips with a large cone angle to 60 K/mW for the narrow long ones. Temperatures of up to approximate to 470 degrees C have been measured close to the aperture with an optical input power of several mW before thermal damage of the coating occurred. The temperature profiles are analyzed theoretically taking into account the optical absorption, the thermal conductivity of the tip, as well as the heat loss to the environment.

Abstract

Abstract

Abstract

The length of the molten zone determines the length of pulled optical fibre tips. Tips produced by laser or filament heating are rather lengthy. By using a foil heater the taper length can be shortened and cone angles in the order of 30 degrees can reproducibly be obtained. For varying the drawing force there is an optimum temperature range where the taper shape is monotonic for the whole tip. The tip end diameter is well below 100 nm for optimized pulling conditions.