C. Tietz

Single spin states in a defect center resolved by optical spectroscopy

Individual paramagnetic defect centers in diamond nanocrystals have been investigated by low-temperature high-resolution optical spectroscopy. Narrow fluorescence excitation spectral lines have been found, indicating transitions between individual spin sublevels. Spectral diffusion is explained by cross relaxation among spin sublevels and by the presence of excited electrons in the conduction band of diamond. The relaxation times are in the millisecond range. The system may be useful for quantum information processing with individual electron spins.

Single Molecule Spectroscopy on the Light-Harvesting Complex II of Higher Plants

Spectroscopic and polarization properties of single light-harvesting complexes of higher plants (LHC-II) were studied at both room temperature and T < 5 K. Monomeric complexes emit roughly linearly polarized fluorescence light thus indicating the existence of only one emitting state. Most probably this observation is explained by efficient triplet quenching restricted to one chlorophyll a (Chl a) molecule or by rather irreversible energy transfer within the pool of Chl a molecules. LHC-II complexes in the trimeric (native) arrangement bleach in a number of steps, suggesting localization of excitations within the monomeric subunits. Interpretation of the fluorescence polarization properties of trimers requires the assumption of transition dipole moments tilted out of the symmetry plane of the complex. Low-temperature fluorescence emission of trimers is characterized by several narrow spectral lines. Even at lowest excitation intensities, we observed considerable spectral diffusion most probably due to low temperature protein dynamics. These results also indicate weak interaction between Chls belonging to different monomeric subunits within the trimer thus leading to a localization of excitations within the monomer. The experimental results demonstrate the feasibility of polarization sensitive studies on single LHC-II complexes and suggest an application for determination of the Chl transition-dipole moment orientations, a key issue in understanding the structure-function relationships.

Spectroscopy of Single N-V Centers in Diamond

Over the past few years, the detection of single N-V centers in diamond has attracted much interest, since it is expected to lead to innovative applications in various domains of quantum information. The N-V center in diamond is a defectconsisting of a substitutional nitrogen atom adjacent to a carbon-atom vacancy. The optical transition between the 3A ground state and the 3E excited state has a very high quantum efficiency allowing single defect spectroscopy. Because of the paramagnetic nature of the ground electronic state, single N-V defects are believed to be promising candidates for solid state quantum computation. To date, however, the photophysics of the defect is not entirely understood. The existence of a singlet metastable state 1A was challenged in a series of papers reporting hole-burning, optically detected magnetic resonance and single molecule experiments. We present here an overview of recent low and room temperature data, indicating the important role of a metastable state of the defect in single center experiments.

Measurement of Stochastic Entropy Production

Using fluorescence spectroscopy we directly measure entropy production of a single two-level system realized experimentally as an optically driven defect center in diamond. We exploit a recent suggestion to define entropy on the level of a single stochastic trajectory [Seifert, Phys. Rev. Lett. 95, 040602 (2005)10.1103/PhysRevLett.95.040602]. Entropy production can then be split into one of the system itself and one of the surrounding medium. We demonstrate that the total entropy production obeys various exact relations for finite time trajectories.

Dual function of cysteine rich domain (CRD) 1 of TNF receptor type 1: Conformational stabilization of CRD2 and control of receptor responsiveness

The proinflammatory cytokine Tumor Necrosis Factor (TNF) exists as a homotrimer, capable of binding three receptor molecules. However, signal competent ligand/receptor complexes form large clusters, likely to be stabilized by additional molecular interactions. Both TNF receptors, TNFR1 and TNFR2, contain four cysteine rich domains (CRD) in their extracellular parts. Previous work showed that the membrane distal CRD1 carries a homophilic interaction domain. Here, we investigated the functional role of CRD1 and its two submodules, A1CRD1 and B2CRD1, in a TNFR1-Fas chimera model system. Removal of CRD1 abolishes TNF binding. In line with these data, molecular dynamics simulations suggest that B2CRD1 of TNFR1 serves as a scaffold to stabilize CRD2 in a conformation necessary for high affinity ligand binding. Deletion of only the N-terminal half of CRD1 (DeltaA1CRD1) of TNFR1 marginally affects ligand binding but abrogates responsiveness towards soluble TNF and reduces effectiveness as a dominant negative inhibitor of wild type TNFR1. A TNFR1-derived molecule containing the CRD1 from TNFR2 also shows reduced responsiveness to soluble TNF. These data strongly suggest that CRD1 is not only crucially involved in multimerization of unligated receptors, but is also directly involved in formation of signal competent ligand/receptor clusters, thereby controlling receptor responsiveness.

Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 x 10(-9) cm(2) s(-1) independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.

Spin-selective low temperature spectroscopy on single molecules with a triplet-triplet optical transition: Application to the NV defect center in diamond

The spin-selective photokinetics of a single matrix-isolated impurity molecule with a triplet-triplet optical transition, T0–T1, is considered and the manifestations of the photokinetics in the fluorescence excitation spectra and intensity autocorrelation functions g(2)(τ) of the molecule undergoing narrow-band optical excitation is studied to resolve the fine structure of the transition. The rates of intersystem crossings (ISCs) T1→S→T0 to and from a nonradiating singlet state S of the molecule and the rate of population relaxation among the ground (T0) state sublevels can be obtained from the spectra and g(2)(τ) using the analytical expressions obtained. New experiments on an individual NV defect center in nanocrystals of diamond, where, for the first time, the fine structure of its triplet-triplet 3A-3E zero-phonon optical transition (~637 nm) at 1.4 K was resolved, are interpreted. It is concluded that the rate of the ISC transition from the mS=0 sublevel of the excited 3E state to the singlet 1A state (~1 kHz) is much slower than the rates from the mS=±1 substates, while the rates of ISC transitions to different mS substates of the ground 3A state are close to each other (~1 Hz). As a result, only the optical transition between mS=0 sublevels in the 3A-3E manifold contributes strongly to the fluorescence. The experimentally observed double-exponential decay of the g(2)(τ) function is explained by the two pathways available to the center for it to leave the S state: (i) the S→ T0(mS)=0) transition and (ii) the S→T0(mS=±1) transitions followed by the slow spin-lattice relaxation T0(mS=±1)→T0(mS=0) (rate ~0.1 Hz). The work is important for studies where the NV center is used as a single photon source or for quantum information processing.

Polarization Measurements on Single Pigment‐Protein Complexes

Individual antenna complexes from different photosynthetic units have been investigated by single molecule spectroscopy. In such energy transfer systems the polarization of the fluorescence emission gives valuable information about the nature of the emitting state, which is not readily available with other methods like fluorescence excitation or emission spectroscopy. The peripheral antenna light harvesting complex from purple bacterium Rhodopseudomonas Acidophila shows predominantly linear polarized fluorescence emission at low temperature, whereas at room temperature the fluorescence is randomly polarized. This is attributed to the fact, that at low temperature in the fluorescence emitting state the excitation energy is localized mainly on 4-5 chromophores. Analysis of the fluorescence emission of single peripheral antenna complexes of green plants indicate that for trimers of this species more than one Chlorophyll is responsible for the final fluorescence emission, which points towards a weak intermonomer coupling in the complex.

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2 x10(-9)cm(2)/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D=3.1 x10(-9)cm(2)/s) with a marked reduction after 30 min of TNF treatment (D=0.9 x 10(-9)cm(2)/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.

Single Molecule Spectroscopy of Oriented Recombinant Trimeric Light Harvesting Complexes of Higher Plants

The bleaching dynamics of reconstituted single light-harvesting chlorophyll a/b investigated. The complexes containing one histidine6 tag per monomeric subunit were immobilised predominantly in a defined orientation with their symmetry axis perpendicular to a Ni-ion-containing surface allowing for the first time the examination of single LHCIIb in an aqueous environment. Most complexes exhibit photobleaching in one step, indicating coupling between the monomeric subunits leading to an energy transfer between adjacent subunits. Differences in bleaching behaviour between these and previous observations with single LHCIIb are discussed.