Cloning and structural basis of fluorescent protein color variants from identical species of sea anemone, Diadumene lineata.

Diadumene lineata is a colorful sea anemone with orange stripe tissue of the body column and plain tentacles with red lines. We subjected Diadumene lineata to expression cloning and obtained genes encoding orange (OFP: DiLiFP561) and red fluorescent proteins (RFPs: DiLiFP570 and DiLiFP571). These proteins formed obligatory tetramers.

Fluorescence microscopic imaging of single desmin intermediate filaments elongated by the presence of divalent cations in vitro.

The formation of intermediate filaments (IFs), a paradigmatic assembly system in biological macromolecules, depends on cations. Herein, to explore the combined effect of ionic strength and divalent cations, we used fluorescence microscopy and examined the in vitro effects of MgCl, CaCl, and SrCl on the KCl concentration-dependent growth of desmin IFs. Fluorescently-labeled desmin IF assembly initiated by KCl and 5 mM divalent cations led to the formation of single desmin IFs in the KCl concentration range of 25-50 mM.

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense.

Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT) next to the first start codon of pRSET.

Adhesion and Disintegration Phenomena on Fractal Agar Gel Surfaces.

In the present study, mechanical phenomena on fractal agar gel were analyzed to understand the interfacial properties of hydrophilic biosurfaces. The evaluation of adhesion strength between the fractal agar gel surfaces showed that the fractal structure inhibits the adhesion between the agar gel surfaces. In addition, when the disintegration behavior of an agar gel block was observed between fractal agar gel substrates, the rough structure prevented the sliding of an agar gel block.

Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release.

Aims: In atrial fibrillation (AF), abnormalities in Ca(2+) release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF.

Methods And Results: Using high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals.

Wetting dynamics of colloidal dispersions on agar gel surfaces.

The effects of silica particle addition on the wetting velocity on flat and fractal agar gel surfaces were analyzed along with the applicability of such particles for controlling the wetting dynamics of water. The contact angles (θD) of the colloidal dispersions obeyed the power law, i.e.

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering.

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors.

Transfection of living HeLa cells with fluorescent poly-cytosine encapsulated Ag nanoclusters.

The fluorescence of silver clusters encapsulated by single stranded oligo-DNA (24 cytosine base pairs, C(24):Ag(n)) was used to monitor the transfection of this new silver/DNA fluorophore inside living HeLa cells. For this, the C(24):Ag(n) molecules were complexed with a commercially available transfection reagent Lipofectamine and the internalization of C(24):Ag(n) was followed with confocal fluorescence microscopy. Bright near-infrared fluorescence was observed from inside the transfected HeLa cells, when exciting with 633 nm excitation, opening up the possibility for the use of these C(24):Ag(n) clusters for biological labelling and imaging of living cells and for monitoring the transfection process with limited harm to the living cells.

Data storage based on photochromic and photoconvertible fluorescent proteins.

The recent discovery of photoconvertible and photoswitchable fluorescent proteins (PCFPs and RSFPs, respectively) that can undergo photoinduced changes of their absorption/emission spectra opened new research possibilities in subdiffraction microscopy and optical data storage. Here we demonstrate the proof-of-principle for read only and rewritable data storage both in 2D and 3D, using PCFPs and RSFPs. The irreversible burning of information was achieved by photoconverting from green to red defined areas in a layer of the PCFP Kaede.

Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores.

We report a rationale for identifying superior dyes for stimulated-emission depletion (STED) microscopy. We compared the dyes pPDI and pTDI, which displayed excellent photostability in single-molecule spectroscopy. Surprisingly, their photostability and performance in STED microscopy differed significantly.

Improved method for counting DNA molecules on biofunctionalized nanoparticles.

In order to accurately determine low numbers (1-100) of immobilized ssDNA molecules at a single, silica 250 nm nanoparticle surface, we hereby propose an integrated approach combining classic single molecule confocal microscopy (SMCM), that is, stepwise photobleaching of labeled ssDNA, with modified total internal reflection fluorescence microscopy (mTIRF). We postulate that SMCM alone is unable to exactly account for all labeled ssDNA because of inherent laser polarization effects; that is, perpendicularly oriented molecules to the sample surface are not (or are only slightly) susceptible to laser excitation and thus are invisible in a classic photobleaching experiment. The SMCM method accounts for at best two-thirds (68%) of the present ssDNA molecules.

Excitation energy migration processes in cyclic porphyrin arrays probed by single molecule spectroscopy.

By using single molecule fluorescence spectroscopy we have investigated the excitation energy migration processes occurring in a series of cyclic porphyrin arrays bearing a close proximity in overall architectures to the LH2 complexes in purple bacterial photosynthetic systems. We have revealed that the conformational heterogeneity induced by the structural flexibility in large cyclic porphyrin arrays, which provides the nonradiative deactivation channels as an energy sink or trap, reduces significantly the energy migration efficiency. Our study provides detailed information on the energy migration efficiency of the artificial light-harvesting arrays at the single molecule level, which will be a guideline for future applications in single molecular photonic devices in the solid state.

Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa.

The fast and reversible on/off switching of the fluorescence emission of the GFP-like fluorescent protein Dronpa has attracted considerable interest for applications in subdiffraction imaging. In this paper we study the use of a donut-mode beam in combination with two more overlapping laser beams to increase the imaging resolution through selective switching to the nonfluorescent photoswitched state. We devise and run a series of numerical simulations to determine suitable photophysical parameters of prospective, thermally stable photoswitchable molecules, in terms of photoswitching quantum yields, fatigue resistance, and possible presence of transient nonfluorescent states.

A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants.

The photophysical properties and photoswitching scheme of the reversible photoswitchable green fluorescent protein-like fluorescent proteins Dronpa-2 and Dronpa-3 were investigated by means of ensemble and single-molecule fluorescence spectroscopy and compared to those of the precursor protein Dronpa. The faster response to light and the faster dark recovery of the new mutants observed in bulk also hold at the single-molecule level. Analysis of the single-molecule traces allows us to extract the efficiencies and rate constants of the pathways involved in the forward and backward switching, and we find important differences when comparing the mutants to Dronpa.

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.

Fluorescence of single molecules in polymer films: sensitivity of blinking to local environment.

The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed.

Orientational effects in the excitation and de-excitation of single molecules interacting with donut-mode laser beams.

The interactions between single molecules and three-dimensional donut modes in fluorescence microscopy are discussed based on the vector diffraction theory of light.We find that the use of donut modes generated from a linearly polarized laser beam can yield information about the orientation of immobilized single molecules, allowing for their use in orientational imaging. While fairly insensitive over a range of orientations, this technique is seen to be very sensitive for the subset of orientations where the transition dipole of the molecule is oriented close to the optical axis of the microscope and perpendicular to the input polarization.

Single-molecule spectroscopic investigation of energy migration processes in cyclic porphyrin arrays.

Covalently linked cyclic porphyrin arrays have been synthesized to mimic natural light-harvesting apparatuses and to investigate the highly efficient energy migration processes occurring in these systems for future applications in molecular photonics. To avoid an ensemble-averaged picture, we performed a single-molecule spectroscopic study on the energy migration processes of cyclic porphyrin arrays and a linear model compound embedded in a rigid polymer matrix by recording fluorescence intensity trajectories, by performing coincidence measurements, and by doing wide-field defocused imaging. Our study demonstrates efficient energy migration within the cyclic porphyrin arrays at the single-molecule level.

Fast and reversible photoswitching of the fluorescent protein dronpa as evidenced by fluorescence correlation spectroscopy.

Controlling molecular properties through photoirradiation holds great promise for its potential for noninvasive and selective manipulation of matter. Photochromism has been observed for several different molecules, including green fluorescent proteins, and recently the discovery of a novel photoswitchable green fluorescent protein called Dronpa was reported. Dronpa displays reversible and highly efficient on/off photoswitching of its fluorescence emission, and reversible switching of immobilized single molecules of Dronpa with response times faster than 20 ms was demonstrated.

The fabrication of a thin, circular polymer film based phase shaper for generating doughnut modes.

We demonstrated the fabrication of a phase shaper for generating a 'doughnut mode' laser beam using a thin, circular polymer film on a substrate. The fabrication method is based on a combination of spin-coating and drop-casting. The alignment procedure to get ideal 'doughnut modes' is described.

Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching.

Recently, reversible photoswitching in bulk samples or in individual molecules of Dronpa, a mutant of a green fluorescent protein (GFP)-like fluorescent protein, has been demonstrated. Intense irradiation at 488 nm changed Dronpa in a dim protonated form, and weak irradiation at 405 nm restored it to the bright deprotonated form. Here, we report on the mechanism of photoswitching of Dronpa by means of ensemble and single-molecule spectroscopy.