The main aim of this study is to investigate correlations between the impact of an external mechanical force on the molecular framework of fluorophores and the resultant changes in their fluorescence properties. Taking into account previous theoretical studies, we designed a suitable custom-tailored oligoparaphenylenevinylene derivative (OPV5) with a twisted molecular backbone. Thin foils made of PVC doped with 100 nM OPV were prepared. By applying uniaxial force, the foils were stretched and three major optical effects were observed simultaneously. First, the fluorescence anisotropy increased, which indicates a reorientation of the fluorophores within the matrix. Second, the fluorescence lifetime decreased by approximately 2.5% (25 ps). Finally, we observed an increase in the emission energy of about 0.2% (corresponding to a blue-shift of 1.2 nm). In addition, analogous measurements with Rhodamine 123 as an inert reference dye showed only minor effects, which can be attributed to matrix effects due to refractive index changes. To relate the observed spectroscopic changes to the underlying changes in molecular properties, quantum-chemical calculations were also performed. Semiempirical methods had to be used because of the size of the OPV5 chromophore. Two conformers of OPV5 (C(2) and C(i) symmetry) were considered and both gave very similar results. Both the observed blue-shift of fluorescence and the reduced lifetime of OPV5 under tensile stress are consistent with the results of the semiempirical calculations. Our study proves the feasibility of fluorescence-based local force probes for polymers under tension. Improved optical sensors of this type should in principle be able to monitor local mechanical stress in transparent samples down to the single-molecule level, which harbors promising applications in polymer science and nanotechnology.
that melt at high temperatures (greater than 80 oC). This function is developed using a three-state model that approximates the ensemble of partially fluorescent premelted states as single state. The utility of this model is demonstrated by the extraction of melting temperatures from a set of synthesized DNA oligomers in both macro-and microscale environments to calibrate laser-heating for microdroplet PCR.
Fluorescence microscopy is a standard tool in molecular biophysics, but even the best resolution obtained by diffraction-limited conventional optical techniques misses the molecular level by two orders of magnitude. In order to overcome the classical diffraction limit, several sub-diffraction resolution imaging methods have been introduced so far. Direct stochastic optical reconstruction microscopy (dSTORM) (1) and PAINT (points accumulation for imaging in nanoscale topography) (2) have the potential to shed light on the intracellular organization of cells with near-molecular resolution. These techniques will be used to localize labelled peptides binding to receptors located in the membrane of protoplasts of the flowering plant Arabidopsis (a model organism for plant research). Binding dynamics will be studied by fluorescence correlation spectroscopy (FCS). References:(1) M.
In a world where more people grow older aging-related neurodegeneration like Alzheimer's disease (AD) affects more and more people. Today, AD can be diagnosed with certainty only post mortem, detecting insoluble b-amyloid peptide (Ab) aggregates and neurofibrillary tangles in the patient's brain tissue. Aggregates consisting of Ab are a fundamental pathologic feature of AD. Today in many studies, concentrations of monomeric Ab in body fluids are investigated, especially for diagnostic purposes. Nevertheless, for the detection, quantitation and qualification of aggregated pathologic Ab forms, also in the course of aging, a highly sensitive detection assay system for aggregated Ab species is necessary. We developed an ultra-sensitive assay for the detection of aggregated protein species out of body fluids. This highly specific and sensitive assay uses confocal fluorescence spectroscopy methods and is sensitive enough to detect single aggregates. For the procedure, pathologic aggregates out of body fluids are immobilized on a glass chip, subsequently fluorescence labeled and detected via confocal spectroscopy. Actually, we are optimizing the assay in concerns of instrumentation (imaging) and microscopy high-resolution and even super-resolution methods. We are developing methods to analyze aggregates via super-resolution microscopy. Setups like PAINT (Point Accumulation for Imaging in Nanoscale Topography) or STORM (Stochastic Optical Reconstruction Microscopy) allow resolutions in nanometer-range. PAINT is based on replacing the point-spreadfunction (PSF) of a fluorophore by a point in the middle of a 2D gaussian fit. First measurements show resolutions of 30 nm. STORM is based on highaccuracy localization of photoswitchable fluorophores. During one imaging cycle, only a small part of the fluorophores is turned on. This allows a high accuracy in determining the fluorophore position by replacing the PSF. The fluorophore positions obtained from a series of imaging cycles can be used to reconstruct the whole image.
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