ChemPhysChem

Intensity fluctuations of individual fluorophores, also termed blinking, are studied at the single-molecule level by T. Cordes, P. Tinnefeld et al. on p. 931. Therefore, dye molecules of the homologous series of cyanines were immobilized through double-stranded DNA on BSA-coated cover slips. The OFF-times due to radical-ion formation increase for larger chromophore size and correlate with redox potentials. Cover artwork by Christoph Hohmann, Nanosystems Initiative Munich (NIM).

Surface-enhanced fluorescence photo-activation localization microscopy reveals imaging of the point-spread-function and spatial distribution of individual fluorescent molecules near densely labeled metal nanostructures as shown by H. Uji-i et al. on p. 973.

What shape is it? Single-molecule and ensemble time-resolved studies support the notion that the π-bond in large macromolecules, such as conjugated polymers, is remarkably persistent in space: even individual chromophores can be bent and twisted, so that caution is warranted when interpreting a wide range of polarization-based spectroscopies.

Blinking and bleaching: Coordination of Mn²⁺ to DNA induces intersystem crossing, causing fluctuations in the fluorescence intensity and accelerated photobleaching.

Molecule-by-molecule arrangement of proteins, for example, in enzymatic networks of predefined composition and proximity, is a major goal that may be accomplished by the single-molecule cut-and-paste technique (SMC&P). For this purpose, co-expressed anchors and handles as protein tags should be employed. As a first step in this direction, the authors develop an SMC&P design which exploits an antibody–peptide complex as a molecular handle.

A “bendy” protein sensor: A DNA-based sensor that uses folded DNA (through DNA kinks) and protein-induced bending to detect DNA-binding proteins is presented. Single-molecule sensing of a transcriptional activator (catabolite activator protein, CAP, which bends its DNA site by 80°) is demonstrated in solution and on surfaces, both in buffers and in cell lysates. The method should allow detection of a wide range of DNA-bending proteins.

Clearly visible: A water-soluble and highly fluorescent perylenediimide is synthesized via ruthenium-catalyzed alkylation with outstanding yields (see scheme). For the first time, the possibility to use phosphonate derivatives in a Murai-type reaction is demonstrated.

High-resolution imaging and spectroscopy of single CdSe nanowires and carbon nanotubes using tip-enhanced near-field optical microscopy increases the optical excitation and emission rates within a nanoscale sample volume. The resulting signal enhancement for Raman scattering and photoluminescence as well as the tip–sample-distance dependence are investigated.

The missing link: Blinking of three dyes from a homologous series (Cy3, Cy5, Cy7) is studied (see picture). The underlying radical anion states are induced by controlling the oxygen concentration and by adding the reductant ascorbic acid. For different conditions the OFF-state lifetime always increases in the order Cy3<Cy5<Cy7. Off-state lifetimes are related to higher reduction potentials as well as chromophore size.

Long time, no see: The long triplet state lifetime of hexa-peri-hexabenzocoronene (blue) appears as dark states in the emission of the perylenemonoimide periphery (green).

Labyrinthine: Quantitative insight into phototphysics of organic dye nanoparticles and the dewetting induced patterns formed from a push–pull triarylamine dye is obtained by using simultaneous absorption and fluorescence microscopy (see picture). The quantum yield of organic nanoparticles and the number of molecules forming the nanoparticles and patterns are deduced.

Influential nanorods: Polarization-dependent enhanced Raman spectra of non-resonant adenine molecules are obtained using single gold nanorods and a parabolic mirror-assisted optical microscope with higher-order laser beams. The influence of the three-dimensional orientation of individual gold nanorods on the Raman enhancement and the nanorod excitation are discussed.

Quenching the glow! The size-dependent photoluminescence quenching of semiconductor quantum dots by functionalised dye molecules in solution is analysed with respect to Förster resonance energy transfer (FRET) and non-FRET contributions (see picture). By this approach, assembly formation and ligand dynamics can be studied at extremely low concentrations of quantum dots and dye molecules.

Maps on metals: Photoactivation localization microscopy (PALM) is applied to study suface-enhanced fluorescence (SEF) on metal nanostructures (SEF-PALM), see picture. The detection of fluorescence from individual single molecules can be used to image the point-spread function and spatial distribution of the fluorescence emitted in the vicinity of a metal surface.

Compensating Mechanisms: The temperature-dependent desorption of single polypeptides from hydrophobic surfaces, studied by means of atomic force microscopy and molecular dynamics simulations, shows a strong compensation mechanism between the different contributions to the desorption force. This is reminiscent of the protein-folding process, where large entropic and enthalpic contributions compensate each other, resulting in a small free-energy difference between the folded and unfolded states.

Watch them glow: A description and theoretical evaluation of dual-focus fluorescence correlation spectroscopy as a tool for measuring diffusion within lipid bilayers is presented (see picture). The method is applied to a study of the dependence of lipid diffusion on lipid and buffer composition with regard to the ionic strength of the buffer, particularly concerning the influence of mono- and divalent ions.

Moving giant vesicles: In a biomimetic assay, giant unilamellar vesicles with membrane-anchored kinesin-1 motor proteins are transported along a two-dimensional microtubule network (see picture). In this assay, cooperatively acting motor proteins enable long traveling distances up to the millimeter range.

3D is here to stay: Three-dimensional super-resolution astigmatic optical imaging provides images of the FtsZ Z-ring in bacterial cells in various stages of the cell cycle. The picture shows, left to right: live stalked cell, live pre-divisional cell, fixed stalked cell, fixed pre-divisional cell. Scale bar: 200 nm.

Dynamic studies: Fluorescence correlation spectroscopy (FCS) data are globally analyzed using the Bayesian inference. This approach is applied, in combination with the results from single-molecule burst analysis, to investigate the influence of various labels on DNA hairpin kinetics as a function of the salt concentration.

Photoinduced isomerization of Cy5 from trans to cis can be quickly reversed with long-wavelength secondary illumination. Modulating this co-illumination directly encodes the modulation waveform on the higher energy Cy5 fluorescence, enabling its selective recovery even in the presence of overwhelming background. This low-energy, reversible, long-lived dark state makes Cy5 an excellent dye for synchronously amplified fluorescence image recovery (SAFIRe).

Let it shine: Gold nanoparticles (see picture) are decorated with an organic fluorophore that possesses fluorescence sensitive to the polarity and hydrogen-bonding properties of the surrounding local environment.

Lifetime and polarization filtered fluorescence correlation spectroscopy, referred to as filtered FCS (fFCS), allows one to study dynamic binding equilibria sensed, for example, by anisotropy in the 2D diagram. The species-specific auto- (SACF) and cross-correlation (SCCF) functions for species (1) and (2) allow one to characterize the equilibrium via the relaxation time for interconversion (gray bar) and the corresponding species fractions (black arrow).

Imaging nucleic acids: Label-free optical imaging of nucleic acids in live cells is demonstrated using highly sensitive stimulated Raman scattering (SRS) microscopy. This technique allows in vivo imaging of DNA condensation associated with cell division.

In one fell swoop: By combining pulsed interleaved excitation with multiparameter fluorescence detection in burst analysis experiments, all information necessary for an accurate spFRET experiment can be collected in a single measurement (see picture for experimental setup). In addition, artifacts can be detected and often corrected.

Complex fluctuation patterns discovered by single-molecule experiments require statistical methods to analyze long multistate hopping traces. The authors give an introduction on how to implement hidden Markov modeling for this purpose and test the method on a calcium binding protein.

Trap and track: The orbital tracking method (OTM) allows us to simultaneously track single nanoparticles (NPs) and spectroscopically characterize their emission while diffusing even inside cells. As the NPs are always trapped by the laser, a detail spectroscopic characterization of the NPs can be done, even if the NPs are moving in three dimensions and along microns (see picture).