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Single Molecules

Wiley Online Library : Single Molecules

Published: 2002-11-01T00:00:00-05:00


Photon Statistics in Single Molecule Experiments


Single molecule fluorescence experiments yield a stream of photocounts, whose statistical properties contain valuable information about processes within the molecule and in its microscopic environment. The photon statistics display features specific of single quantum systems and are therefore best discussed in the frame of a quantum mechanical theory of radiation. The present summary of a lecture given in April 2002 at the Hofgeismar Spring School on single molecules presents the main concepts used in quantum theory of light, together with a few useful references, and discusses some illustrations and applications to single molecule measurements drawn from the recent literature.

Single Protein Molecules Visualized and Tracked in the Interior of Eukaryotic Cells


In the last few years the visualization and tracking of single fluorescent proteins, nanometer-sized RNP particles and viruses within the cellular interior was accomplished. This became feasible by use of photostable fluorescent dyes, extremely low probe concentrations, and wide-field fluorescence microscopic setups equipped with sensitive slow-scan or intensified CCD cameras. This paper reviews the results of the studies performed so far, discusses potential problems and gives an outlook on future applications.

DNA-based Molecular Nanotechnology


The use of molecular building blocks opens a new dimension for nanotechnology. Biomolecules offer a variety of possibilities for manipulation, provide a new size dimension and are especially suitable for “bottom up” approaches. Nucleic acids are of special interest due to their ability of self-organization, the achieved combinatorial information capacity and its molecular-biological processability. Here we present an approach for a molecular component systems with DNA-based elements and products that is suitable for molecular nanotechnology. Oligonucleotides thereby serve as biological modifiers of nanoparticles and surfaces to form self-assembling monolayers, and genomic DNA acts as framework for the building blocks. A first application of DNA-nanoparticle complexes could be the use as a novel, highly-stable label for chip technologies, with the potential for single-molecule detection. Another field is the fabrication of novel electronic devices, based on extreme miniaturization. This paper describes the different fields of use for DNA-based molecular modules, and presents first results of the realization of this concept.

On the Field Enhancement at Laser-illuminated Scanning Probe Tips


First results of a new dynamic approach to calculate the field enhancement at a metal ellipsoid close to a metal surface are presented. The theoretical approach is based on the solution of Maxwells equations. The numerical calculation is influenced by the dielectric constants of both metals, the angle of incidence of the monochromatic electromagnetic wave, the distance of the ellipsoid to the metal surface and the semi axis ratio of the ellipsoid.

Surface-enhanced and STM-tip-enhanced Raman Spectroscopy at Metal Surfaces


The strong electromagnetic field enhancement, occurring at illuminated metal structures with sub-wavelength dimensions, is exploited to develop Raman spectroscopy with exceedingly high sensitivity and lateral resolution, possibly down to single molecule detection. After reviewing Surface Enhanced Raman Spectroscopy (SERS) on single crystalline surfaces and colloids we present recent results on the Tip Enhanced Raman Spectroscopy (TERS) approach, where a metal tip is used as an external enhancing unit. In this way the electromagnetic and chemical surface enhancement are physically separated: the former is confined to the tip, the latter to the metal-adsorbate system. So far, TERS has been reported only for a few molecules exhibiting large Raman cross sections, such as sulphur or dye molecules. Here, we present combined SERS and TERS studies for the CN- ions and TERS for Brilliant Cresyl Blue adsorbed at smooth thin gold films.

STM Investigation on Single, Physisorbed Dendrimers


Porphyrin dendrimers were synthesized to mimic naturally occurring proteins, which catalyze a number of biochemically important reactions. In addition, chiral dendrimers were prepared as model compounds for the study of nanoscopic chirality. The structures of these dendrimers cannot be characterized by x-ray, as most dendrimers do not crystallize. We succeeded to image single, physisorbed dendrimers on noble metal surfaces with STM. All examined dendrimers can easily be (re)moved with the STM tip, even at low scanning currents (low pA range). One possibility to avoid this, is to change the peripheral groups of the dendrimer, so that they chemisorb on the surface. We decided to study physisorbed macromolecules with unchanged molecular properties, and investigated how the approach by Tokuhisa et al. for chemisorbed polyamidoamin (PAMAM) dendrimers, could be adapted to our molecules. Indeed, even physisorbed molecules are stabilized by embedding them in a self-assembled monolayer of covalently bonded 1-hexadecanethiol. The so embedded dendrimers show a reduced mobility on the substrate with a conformation closer to the one in solution, permitting STM imaging at various temperatures.

The Imaging of Small Domains of J-Aggregated Dye Molecules by Scanning Near-Field Optical Microscopy


The concept of high resolution imaging of a sample on a metal substrate by scanning near field optical microscopy (SNOM) with the tetrahedral tip is used for imaging of domains of oriented dye molecules on a metal substrate at a resolution in the order of 10 nm. The metal substrate is important for obtaining a high resolution in the SNOM images and at the same time it is expected to provide a mechanism for an increased contrast in SNOM imaging by scanning probe enhanced elastic scattering of molecules in the gap between a metal tip and a metal surface.

Theory of Near-field Optical Imaging with a Single Molecule as Light Source


Scanning near-field optical microscopes (SNOM) illuminate a sample in the very near-field using a nanometer sized tip. Ideally, the light source should be point-like and many efforts have been made to optimize tip efficiency (see, for example, the article of Heimel et al in this issue). Very recently, Sandoghdar et al have realized a molecular probe tip in which a terrylene molecule inserted in a paraterphenyl microcrystal is attached at the extremity of the probe tip [1]. The excited molecule behaves as a point-like light source which is raster scanned over an aluminium patterned structure. We propose here an analysis of this experiment based on the field-susceptibility formalism (also called Green's Dyadic Method) [2,3]. In particular, in strong analogy with the Scanning Tunneling Microscope (STM), we will show that the detected signal is proportional to the Local Density of photonic States (LDOS) available at the immediate proximity of the sample.The molecular light source behaves as a dipole p(t), located at rtip and oscillating at the wavelength λ0=2π/ω0=630 nm$$ {\rm p(t) = p_0 \cos (\omega _0 t)} {\bf u}, (1) $$where u is a unit vector that gives the dipole orientation.The electric field E(r,t) created at the point r below the surface by the dipole p(t) is evaluated thanks to the field-susceptibility S(r,rtip,ω) associated to the experimental geometry$$ {\bf E}({\bf r},{\rm t}) = {\bf S}({\bf r},{\bf r} _{\rm tip},\omega _0).{\rm p}_0 {\rm cos} (\omega _0 {\rm t}) {\bf u}. (2)$$Then, the detected signal is the intensity scattered in the solid angle Ω below the surface (see figure 1)$$ {\rm F}(\Omega, {\bf r} _{\rm tip}) = {\int}_{\omega} {\bf E}^2 ({\bf r'},{\rm t}) {\rm d} {\bf S}. (3) $$Additionally, we demonstrate that for large angle of detection, this signal is directly proportional to the partial LDOS nu(rtip,ω0) [2,3]$$ {\rm F} (\Omega = 4 \pi, {\bf r} _{\rm tip}) \propto {\rm n} _{\bf u} ({\bf r} _{\rm tip}, \omega _0), (4) $$where the partial LDOS nu(rtip,ω0) depends on the orientation u as following [3]$$ {\rm n} _{\bf u} ({\bf r} _{\rm tip}, \omega _0) = 1/2 \pi\omega _0 \, {\rm Im} \, {\bf u}. \, {\bf S} ({\bf r} _{\rm tip}, {\bf r} _{\rm tip}, \omega_0). {\bf u} (5) $$We also propose a simple interpretation of the relationship between the LDOS and the detected signal in strong analogy with the STM [3]. This analogy can be made thanks to the equivalency between the illuminating mode SNOM and a particular collecting mode configuration [3,4]. Finally, the experiment realized by Sandoghdar et al is analysed both numerically and theoretically [1,3]. Moreover, the concept of optical corrals is introduced [5].

Plug Harvesting by Atomic Force Microscopy


Aldosterone is an important mediator of osmoregulation in vertebrate cells and is thought to be involved in cell differentation. Its signaling pathway starts with the formation of a complex with the mineralocorticoid receptor (MCR) which is then translocated into the cell nucleus where transcription of target genes is initiated. Finally, mRNAs of specifically activated genes appear in the cytoplasm. Using atomic force microscopy (AFM), we were able to visualize the signaling cascade described above. We injected stage VI oocytes of Xenopus laevis with aldosterone and isolated their nuclei after different incubation times. Nuclei obtained 2 min after application of aldosterone clearly exhibited macromolecules (“flags”) attached to the nuclear pore complexes (NPCs) when examined by AFM. The estimated size of the observed particles (80-160 kDa) corresponds with the actual size of the MCR (∼120kDa). NPCs of oocytes injected about 20 min prior to preparation showed large macromolecules (“plugs”) within their central channels. We concluded that these plugs contain transcripts induced by aldosterone. Electrical measurements support these findings; appearance of both flags and plugs is linked to an increase in nuclear envelope electrical resistance (NEER), probably due to partial plugging of NPCs. The increase of NEER parallel to the occurrence of plugs is prevented by adding RNase A or actinomycin D (an inhibitor of transcription). Alterations of NEER and formation of flags and plugs in response to aldosterone are suppressed by coinjection of the MCR-inhibitor spironolactone. Therefore we consider plugs to represent the early genomic response to aldosterone stimulation of Xenopus oocytes.In conclusion, AFM not only allows imaging of structures in the submicrometer range, it may also be used to manipulate them. By applying forces to the AFM tip approximately 10 fold higher than those used for imaging we were able to dislocate plugs from NPCs (see figures). The macromolecules sticking to the AFM tip can be used as substrates for further experiments, for example RT-PCR protocols.

Influence of Monovalent Salt on the Molecular Structure of Single DNA Complexes with Positively Charged Dendronized Polymers


In cells and viruses as well as non-viral gene delivery systems, DNA is complexed with different molecules to form highly condensed structures. A wide range of conditions that cause DNA to collapse into compact structures has been discovered [1]. However, in most of these cases an exact description of these structures cannot be given. Since the complex stability is largely due to electrostatic forces, it can be modulated by varying the salt concentration. Apart from the biological aspects the study of the molecular structure of polyelectrolyte complexes may be used to improve our general understanding of polyelectrolyte interactions. Theoretical models reveal the structure of complexes formed between a stiff charged cylinder and an oppositely charged flexible or semiflexible polymer [2], [3]. Here we determine the influence of salt on the structure of linearized pUC19 plasmid DNA and positively charged dendronized polymers of generation two (PG2).The repeat units of the polymers are styrenes functionalized with dendrons carrying protonated amine groups at the periphery. Starting with the amino-terminated dendronized polystyrene of generation one (PG1), higher generations were obtained by the so called mixed “attach-to” approach [4]. For the analysis of the structure of the polyelectrolyte complexes scanning force microscopy (SFM) was used. The molecules were allowed to adsorb from solution onto mica or poly-L-ornithin coated mica, rinsed three times with water and finally dried under a stream of N2. Further details of sample preparation are given elsewhere [5]. Complexes of DNA and dendronized polymers of generation two, deposited from different NaCl solutions (10, 50, 100 and 300 mM) onto poly-L-ornithin coated mica, were visualized via SFM (Fig. 1). For the analysis of the complexes the heights and the contour lengths of both the complex (LC) and of the DNA that belonged to the complex (LDNA-C) were determined. While for DNA/PG2 complexes in 0 mM NaCl the average height was (4.0 ± 0.3) nm [5], within the errors the same heights were obtained for the complex using different NaCl solutions (4.2 ± 0.4) nm. The underestimation of the height of molecules in SFM images due to tip-sample interactions (deformation of the sample) is a well known feature. To evaluate the contour lengths of the complex and DNA, their contour was divided into straight segments of 2-5 nm. For their analysis only those complexes were chosen which exhibited a constant height along their contour, and where single DNA strands that came out of this complex belonged clearly to the complex (see [5]). The length of the DNA that contributes to the complex (LDNA-C) can be obtained by subtracting the measured contour length of the DNA molecule out of the complex (Lout) from the length of the monodisperse DNA (L0) by LDNA-C = L0-Lout. In Fig. 2 the contour lengths of the complexes (Lc) are plotted versus the DNA that contributes to the complex (LDNA-C) [5]. In the presence of elevated salt concentration, the obtained data for each NaCl concentration exhibit an overall linear dependence where the slope (m) decreased for increasing concentration of NaCl. Using the estimated radius for PG2 (1.6 ± 10 %) [5] and the theoretical diameter for DNA (2 nm), we calculate the DNA length required for one turn around the dendronized polymers (U) to be (16.3 ± 1.0). With Xi=miU (i stands for the different salt concentrations), the pitch (X) of the wrapped DNA can be calculated. Comparing the results to the case of 0 mM NaCl obtained previously [5], the increase in NaCl concentration lead to a decrease in the pitch separation of DNA which is consistent with the theory [3]. We propose a molecular level structural model for a DNA/dendronized polymer complex, according to which the polyelectrolyte with[...]

Non-exponential Kinetics of Photoblinking and Photobleaching of Rhodamine 6G in Polyvinylalcohol


Photobleaching and photoblinking have proven to be the main bottleneck for single-molecule microscopy and spectroscopy at room temperature. Here, a quantitative ensemble study of the kinetics of photoblinking and photobleaching at room temperature of a typical fluorescent label, Rhodamine 6G, in a polar, hydrogen bonding, solid matrix of polyvinylalcohol is presented as a function of the excitation intensity and the presence of oxygen. To achieve uniform irradiation of all molecules present in the excitation focus, the sample (2.0 x 10–5 M R6G in PVA spin-coated on a quartz substrate) is covered by a pinhole array mask with holes of diameter 40 ∝m, each addressable as an individual sample. The experiments are performed at intensities between 65 mW/cm2 and 320 W/cm2 and the measured emissivity of the system is normalized to that at 65 mW/cm2. The emissivity is shown to decrease by a factor of up to 20 at high intensity indicating the presence of a dark state, which would lead to photoblinking of single molecules. The triplet state of R6G cannot be this dark state, as it is hardly populated at excitation intensities below 1 kW/cm2. However, our data suggest that this state might be an intermediate between the singlet excited state and the dark state, which could be for instance a radical. Fig. 1 shows long-term fluorescence traces obtained at room temperature in an air atmosphere, displaying photobleaching. The rates governing blinking and bleaching are found to be widely distributed. Bleaching is shown to be more efficient in air, while blinking is more pronounced in the nitrogen atmosphere, because the lifetime of the dark state becomes longer.

Individual LH3 (B800-820) Light-Harvesting Complexes Studied by Optical Single-Molecule Spectroscopy


The initial event in bacterial photosynthesis is the absorption of sunlight by an array of pigment-protein complexes, the so called light-harvesting complexes (LH). The absorbed light energy is then efficiently transferred to the reaction centre (RC), where the charge separation and thus the primary conversion into chemical energy takes place. The photosynthetic purple bacterium Rhodopseudomonas (Rps.) acidophila usually contains two types of light-harvesting complexes, light-harvesting complex 1 (LH1) and light-harvesting complex 2 (LH2). When grown under low-light and/or low temperature conditions, an additional spectroscopic variant of LH2 is expressed: the light-harvesting complex 3 (LH3) [1, 2]. The high-resolution crystal structure of LH3, which is also denoted as B800-820, has recently been resolved and revealed a C9-symmetry similar to that of LH2, thus with the bacteriochlorophyll a (BChl a) pigments arranged in two concentric rings. The outer ring of LH3 has nine well-separated BChl a pigments, that absorb at around 800 nm (B800 ring), while the eighteen closely interacting BChl a pigments in the inner ring absorb at around 820 nm (B820 ring). The B800 and B820 pigments are arranged with their molecular plane perpendicular and parallel to the symmetry axis of the complex, respectively. Since the overall structures of LH2 and LH3 are very similar, the interesting question to address is what causes the shift of the long-wavelength absorption band from 850 nm in LH2 down to 820 nm in LH3.It has previously been demonstrated that optical single-molecule spectroscopy at low temperature provides direct insight into relevant parameters determining the electronic structure of LH2 and LH1 complexes [5-8]. Measuring fluorescence-excitation spectra of single LH complexes avoids ensemble averaging and resolves bands otherwise masked by inhomogeneous line broadening. By applying this technique to individual LH3 complexes of Rps. acidophila (str. 7750), we were able to gain more insight into the electronic structure of LH3 as well as in the possible origin of the spectral shift from 850 to 820 nm.The spectra revealed a clear difference in the spectral features of the B800 and B820 band. In the B800 band several, relatively narrow, lines with bandwidths ranging from 2 to 13 cm-1 full width half maximum (FWHM) were observed, whereas in the B820 band only a limited number of broad bands with spectral widths of 60 to 150 cm-1 FWHM were present. These results clearly indicate a difference in electronic structure and dynamics of the bands. In the B800 band the excitations are mainly localized on individual pigments with picosecond dynamics within the band. In the B820 band the excitations are strongly delocalized over the ring with exciton relaxation in the femtosecond regime. Furthermore, studying the polarization dependence of the spectra revealed that about 60 % of the studied complexes show two broad, orthogonally polarized bands in the 820 nm region. These two bands were assigned to the kcirc= ±1 states of a circular exciton which have their degeneracy lifted by δE ± 1 = 160 cm-1. The overall spectral behaviour of the studied LH3 complexes and the previously studied LH2 complexes [5-7] is very similar, even though LH3 exhibits more spectral heterogeneity than LH2.Taking these spectral similarities into account, especially with respect to the excitonic behaviour of the B820 (LH3) and B850 (LH2) bands, it is concluded that the spectral shift from 850 to 820 nm is not caused by changes in the interaction energy. Instead, the spectral shift appears to be induced by changes in the site energies of the pigments.

Inelastic Effects in Molecular Conductors


In this work we present a framework for the calculation of the conduction properties of a metal-molecule-metal junction which is in contact with its thermal environment. The effects of thermal relaxation and dephasing on the transmission properties of the junction were studied using a simple tight binding model for the molecular conductor. The interaction between the molecular system and the thermal environment is described on the level of the Redfield theory, which is a weak electron-phonon coupling scheme, modified for the description of steady-state situations. We show that the transmitted flux consists of two (generally non-separable) components: a flux associated with the elastic tunneling and a thermally activated flux component. The coherent (tunneling) component dominates the transport at low temperatures, large energy gaps and short molecular chains. The incoherent (activated) component is important in the opposite limits. The integrated transmission provides a generalization of the Landauer conduction formula in the presence of thermal relaxation. [1] Using the same formalism, we investigate the issue of heat release on a current carrying molecule: the total amount of heat that is generated on the wire during electrical conduction. Local aspects of the heat release were investigated as well [2]. We compare quantum to classical calculations in the resonance regime, and far from it. For the quantum case we calculate the fraction of the available energy, i.e. of the potential drop, that is converted to heat on the molecular barrier and its dependence on the system parameters: the potential bias, molecule length, dephasing rate and temperature. We find that in the localization limit, where the electron is fully thermalized at each molecular site, all the available energy is dissipated on the bridge, while for short systems and weak system-bath coupling, only a small fraction of the available energy is deposited as heat on the bridge. In our simulations we got this fraction to be of the order 0.1-0.3 using a reasonable range of parameters.We also present a scheme for the analysis of local aspects of heat release, and use it to study the position dependence of the power dissipation for a specific molecular structure.Finally, using classical heat conduction theory we estimate the temperature rise on the molecule due to the heating effects. We find that, within a reasonable range of voltage and molecular parameters, it is in the few degrees range, and therefore it should not affect the molecular junction functionality. It should be emphasized that classical heat transfer theory overestimates heat conduction, so a quantum treatment of vibrational energy transmission in molecular junctions is needed in order to better estimate this temperature rise of the molecular junction. Such study is currently underway.

Job Offers: Single Mol. 5–6/2002