Fluorescent proteins: shine on, you crazy diamond.

In this Perspective we discuss recent trends in the development and applications of fluorescent proteins. We start by providing a historical and structural perspective of their spectroscopic and structural aspects and describe how these properties have made fluorescent proteins essential as 'smart labels' for biosensing and advanced fluorescence imaging. We show that the strong link between the spectroscopic properties and protein structure and properties is a necessary element in these developments and that this dependence makes the proteins excellent model systems for a variety of fields.

Three-dimensional visualization of defects formed during the synthesis of metal-organic frameworks: a fluorescence microscopy study.

Imperfections in the spotlight: fluorescence microscopy was used to detect defects in metal-organic frameworks formed during synthesis. In contrast to currently available techniques, confocal fluorescence microscopy offers the advantage of three-dimensional imaging at the single-crystal level combined with the sensitivity required to study the start of defect formation.

Energy transfer pathways in a rylene-based Triad.

Herein, a study on the excitation energy migration is reported for a newly synthesized Triad with a well-defined architecture consisting of a central terrylenediimide decorated with four perylenediimide and sixteen naphthalenemonoimide chromophores. Steady-state femto- and picosecond time-resolved spectroscopy were employed to unveil the excited states dynamics in solution. Compared with the results obtained from the corresponding model compounds, the Triad is an efficient light collector over the entire visible spectral range and the fluorescence occurs mostly from the core with a quantum yield as high as 60%.

Unraveling excited-state dynamics in a polyfluorene-perylenediimide copolymer.

Insight into the exciton dynamics occurring in a polyfluorene-perylenediimide (PF-PDI) copolymer with a reaction mixture ratio of 100 fluorene units to 1 N,N'-bis(phenyl)-1,6,7,12-tetra(p-tert-octylphenoxy)-perylene-3,4,9,10-tetracarboxylic acid diimide (PDI) is presented here. Time-correlated single photon counting and femtosecond transient absorption spectroscopy measurements on the PF-PDI copolymer have been employed to investigate the excited-state properties of the polyfluorene subunit where the exciton is localized (PF) and the incorporated PDI chromophore. The experimental results were compared with those obtained from a polyfluorene polymer (model PF) and a N,N'-bis(2,6-diisopropylphenyl)-1,6,7,12-tetra(p-tert-octylphenoxy)-perylene-3,4,9,10-tetracarboxylic acid diimide (model PDI) which were used as reference compounds.

Synthesis and photophysics of core-substituted naphthalene diimides: fluorophores for single molecule applications.

The synthesis and photophysics of two new aminopropenyl naphthalene diimide (SANDI) dyes are reported. A general and convenient method for the synthesis of the precursor mono-, di-, and tetrabrominated 1,4,5,8-naphthalene tetracarboxylic dianhydrides is described. The two core-substituted SANDIs exhibit many of the photophysical properties required for fluorescence labeling applications including high photostability and high fluorescence quantum yields (>0.

Characterization of fluorescence in heat-treated silver-exchanged zeolites.

Thermal treatment of Ag(+)-exchanged zeolites yields discrete highly photostable luminescent clusters without formation of metallic nanoparticles. Different types of emitters with characteristic luminescence colors are observed, depending on the nature of the cocation, the amount of exchanged silver, and the host topology. The dominant emission bands in LTA samples are situated around 550 and 690 nm for the samples with, respectively, low and high silver content, while in FAU-type materials only a broad band around 550 nm is observed, regardless of the degree of exchange.

Polymers and single molecule fluorescence spectroscopy, what can we learn?

This tutorial review summarizes the most important results and developments in the field of polymer science by means of single molecule fluorescence spectroscopy (SMFS) at ambient temperatures. A broad range of topics will be addressed and it will be discussed which single molecule methods are suitable to get the maximum amount of information about polymer structure, polymer dynamics and the photophysics of incorporated or embedded dye molecules. In particular, we will report on the use of polymer films for immobilization of molecules, the visualization of dynamics near the glass transition temperature Tg, the reptation of polymer chains, the conformation adopted by polymer chains and the in situ observation of the polymerization reaction itself.

Linking phospholipase mobility to activity by single-molecule wide-field microscopy.

Many of the biological processes taking place in cells are mediated by enzymatic reactions occurring in the cell membrane. Understanding interfacial enzymatic catalysis is therefore crucial to the understanding of cellular function. Unfortunately, a full picture of the overall mechanism of interfacial enzymatic catalysis, and particularly the important diffusion processes therein, remains unresolved.

Giant molecular spoked wheels in giant voids: two-dimensional molecular self-assembly goes big.

We present here the formation of giant pores in surface-confined molecular networks of a triangular-shaped dehydrobenzo-[12]annulene derivative: the diameter of the pores reaches over 7 nm and the giant pores are used as templates to accommodate a giant molecular spoked wheel, which allows us to observe rotational and adsorption-desorption dynamics of single guest molecules.

Guiding the self-assembly of a second-generation polyphenylene dendrimer into well-defined patterns.

A second-generation polyphenylene dendrimer 1 is shown to self-assemble into nanofibers. To guide the formation of the dendrimer fibers into well-defined patterns, 1H,1H,2H,2H-perfluorodecyltrichlorosilane is grafted in the gas phase onto a silicon substrate. De-wetting of the solution on the nanopatterned surface results in the formation of a nanostructured template, into which fiber growth subsequently occurs under the constraints set by the de-wetted morphology.

Molecular clusters in two-dimensional surface-confined nanoporous molecular networks: structure, rigidity, and dynamics.

The self-assembly of a series of hexadehydrotribenzo[12]annulene (DBA) derivatives has been investigated by scanning tunneling microscopy (STM) at the liquid/solid interface in the absence and presence of nanographene guests. In the absence of appropriate guest molecules, DBA derivatives with short alkoxy chains form two-dimensional (2D) porous honeycomb type patterns, whereas those with long alkoxy chains form predominantly dense-packed linear type patterns. Added nanographene molecules adsorb in the pores of the existing 2D porous honeycomb type patterns or, more interestingly, they even convert the guest-free dense-packed linear-type patterns into guest-containing 2D porous honeycomb type patterns.

Photoinduced electron-transfer in perylenediimide triphenylamine-based dendrimers: single photon timing and femtosecond transient absorption spectroscopy.

The excited state dynamics of two generations perylenediimide chromophores substituted in the bay area with dendritic branches bearing triphenylamine units as well as those of the respective reference compounds are investigated. Using single photon timing and multi-pulse femtosecond transient absorption experiments a direct proof of a reversible charge transfer occurring from the peripheral triphenylamine to the electron acceptor perylenediimide core is revealed. Femtosecond pump-dump-probe experiments provide evidence for the ground state dynamics by populating excited vibronic levels.

Site-selective guest inclusion in molecular networks of butadiyne-bridged pyridino and benzeno square macrocycles on a surface.

We present here the formation of a modular 2D molecular network composed of two different types of square-shaped butadiyne-bridged macrocycles, having intrinsic molecular voids, aligned alternately at the solid-liquid interface. Site-selective inclusion of a guest cation took place at every other molecular void in the molecular network with two different recognition sites.

Structural, photophysical, and photochemical characterization of 9-anthracenecarboxylate-hydrotalcite nanocomposites: evidence of a reversible light-driven reaction.

ZnAl hydrotalcites containing increasing amounts of 9-anthracenecarboxylate anion (9AC) have been obtained via an anion-exchange procedure. In particular, intercalated and/or surface-exchanged samples were prepared to study the effect of the chromophore packing on their photophysical and photochemical behavior. Surface-exchanged samples were obtained by equilibrating the carbonate form of the ZnAl hydrotalcite with dilute solutions of 9AC.

Complexation of lipofectamine and cholesterol-modified DNA sequences studied by single-molecule fluorescence techniques.

Lipoplex formation for normal and cholesterol-modified oligonucleotides is investigated by fluorescence correlation spectroscopy (FCS). To overcome the problems related to the fitting of autocorrelation curves when fluorescence bursts are present, the baseline fluorescence levels and the fluorescence bursts in the same trace were separately analyzed. This approach was not previously used in FCS studies of lipoplexes and allowed a more detailed characterization of this heterogeneous system.

Synthesis of dehydrobenzo[18]annulene derivatives and formation of self-assembled monolayers: implications of core size on alkyl chain interdigitation.

The synthesis of a series of dodecadehydrotribenzo[18]annulene ([18]DBA) derivatives is reported, together with their steady-state absorption and fluorescence properties. The main focus, though, is on the self-assembly of these compounds at the liquid-solid interface as investigated with scanning tunneling microscopy (STM), highlighting the effect of alkyl chain orientation and alkyl chain length on the molecular ordering. Owing to the large triangular pi-electron system of the [18]DBAs, two different types of alkyl chain orientation are observed.

Single-molecule fluorescence spectroscopy in (bio)catalysis.

The ever-improving time and space resolution and molecular detection sensitivity of fluorescence microscopy offer unique opportunities to deepen our insights into the function of chemical and biological catalysts. Because single-molecule microscopy allows for counting the turnover events one by one, one can map the distribution of the catalytic activities of different sites in solid heterogeneous catalysts, or one can study time-dependent activity fluctuations of individual sites in enzymes or chemical catalysts. By experimentally monitoring individuals rather than populations, the origin of complex behavior, e.

Visualization of membrane rafts using a perylene monoimide derivative and fluorescence lifetime imaging.

A new membrane probe, based on the perylene imide chromophore, with excellent photophysical properties (high absorption coefficient, quantum yield (QY) approximately 1, high photostability) and excited in the visible domain is proposed for the study of membrane rafts. Visualization of separation between the liquid-ordered (Lo) and the liquid-disordered (Ld) phases can be achieved in artificial membranes by fluorescence lifetime imaging due to the different decay times of the membrane probe in the two phases. Rafts on micrometer-scale in cell membranes due to cellular activation can also be observed by this method.

Single molecule fluorescence spectroscopy of pH sensitive oligonucleotide switches.

Several authors demonstrated that an oligonucleotide based pH-sensitive construct can act as a switch between an open and a closed state by changing the pH. To validate this process, specially designed fluorescence dye-quencher substituted oligonucleotide constructs were developed to probe the switching between these two states. This paper reports on bulk and single molecule fluorescence investigations of a duplex-triplex pH sensitive oligonucleotide switch.

Singlet-singlet annihilation leading to a charge-transfer intermediate in chromophore-end-capped pentaphenylenes.

The excited-state properties of two peryleneimide chromophore end-capped pentaphenylene compounds were investigated in detail using femtosecond transient absorption and single-photon timing experiments. Singlet-singlet annihilation was found to promote one chromophore into a higher excited state and results in the formation of an ultra-short-living intermediate charge-transfer (CT) state in the S(n)-S(1) deactivation pathway. In low-polarity solvents, this CT state is found to be energetically higher than the first excited state and thus cannot be populated via one-photon excitation.

Non-conjugated, phenyl assisted coupling in through bond electron transfer in a perylenemonoimide-triphenylamine system.

Two donor-bridge-acceptor compounds containing triphenylamine (TPA) donors and perylenemonoimide (PMI) acceptors have been studied by spectroscopic techniques and quantum chemical computation. Both systems have been observed to emit prompt and delayed fluorescence under certain conditions indicating that forward and reverse electron transfer (ET) processes can occur between the locally excited and the charge separated states. The experimental and computational results show that the TPA and PMI chromophores are better coupled by almost 50% in the meta isomers which undergo ET more readily than the para isomers.