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Dimethyl-pepep (D-pepep), a newly developed and very efficient two-photon absorber, has been tested here for two-photon excitation (TPE) cellular imaging. The spectral characteristics of the dye following one-photon excitation (OPE) and TPE (excitation and emission spectra, fluorescence lifetime, molecular brightness, saturation intensity) are reported. In vitro interaction studies with biomolecules show that dimethyl-pepep has a large affinity for DNA. A comparison with a widely used DNA stainer, 4-6-diamidino-2-phenylindole (DAPI) bound to DNA shows that the D-pepep brightness is one order of magnitude higher than that of DAPI, making this dye suitable for microscopy and imaging applications. TPE images taken from double-stained yeast Saccharomyces cerevisiae cells have revealed that D-pepep localizes mainly in the nucleus, similarly to DAPI, and in mitochondria, although to a minor extent. Preliminary tests have shown that the dye cellular toxicity is negligible.

Fluorescence anisotropy decay spectroscopy is a suitable tool for investigating the size and the shape of biological molecules. We coupled this technique to an optical microscope in order to reduce the excitation volume and to allow its application to spatially inhomogeneous samples. Phase modulated measurements of the fluorescence anisotropy decay were performed by feeding an intensity modulated linearly polarized laser beam to the epifluorescence port of a microscope. Here we report the test of the dynamic response of the microscope by comparing the lifetime and fluorescence polarization anisotropy decays obtained in cuvettes in a standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor for the objective depolarization is introduced in the best-fit functions for the data analysis of the decays, the results obtained on the two setups are comparable. Some applications are reported here on long DNA tracts as well on short DNA fragments containing structural anomalies.

This report deals with the fundamental quantum physics behind two-photon excitation also providing a link to the experimental consequences exploited in microscopy. The optical sectioning effect is demonstrated as well as the distribution of excitation and of fluorescence emission., (Copyright 2003 Wiley-Liss, Inc.)

Dilute solutions of DNA plasmid pUC18 (2687 base pairs) have been studied by polarized photon correlation spectroscopy. The autocorrelation functions of the scattered light have been measured and fitted to a sum of exponentials. When a two exponential fit of the autocorrelation functions is performed, the slower relaxation rate corresponds to the expected translational diffusion coefficient of the plasmid while the faster one shows a linear behavior below K2[bar_over_tilde:_approx._equal_to]5×1014 m-2, being superlinear at larger K2 values. The translational diffusion coefficient at T=25° is Dt=5.4±0.25×10-12 m2/s while the extrapolation of the fast relaxation rate to K2=0 yields the tumbling rotational coefficient R⊥=1.5±0.2 kHz. Monte Carlo simulations of the plasmid conformation have been performed with different sets of dynamical parameters (bending and torsional rigidities, Debye length), and the hydrodynamic coefficients have been computed according to known algorithms. The translational diffusion coefficient determined experimentally shows good agreement with the results of Monte Carlo simulations, while the simulated rotational tumbling coefficient is slightly larger than that found experimentally. © 1996 American Institute of Physics. [ABSTRACT FROM AUTHOR]

The conformation and slow internal motions of a DNA plasmid molecule pUC18 (2687 base pairs) are studied by means of depolarized photon correlation spectroscopy. The autocorrelation functions are measured at different scattering angles between 6.7° and 90° and analyzed as heterodyne autocorrelation functions. One obtains both a relaxation time of [bar_over_tilde:_approx._equal_to]140 μs, which is interpreted as the tumbling rotational diffusion coefficient of the plasmid, and some faster component with relaxation time around 15 μs. The value of the rotational diffusion relaxation time is in good agreement with the results of Monte Carlo simulations, while the faster decay has a nonsingle exponential decay behavior. The results of measurements made in the depolarized scattering configuration are compared to those obtained from polarized configuration and critically discussed. © 1996 American Institute of Physics. [ABSTRACT FROM AUTHOR]

The dependence of the spinning diffusion coefficient of a wormlike chain upon contour length L, persistence length P, and radius R is shown here to follow a ‘‘Lorentzian’’ law of width Γ vs σβL/R, where σ2β=l0/P is the variance of the bending angles distribution of Monte Carlo simulated chains with bond length l0. This description is equivalent to that of a spinning cylinder of length L and effective radius Reff=R(L,P), with Reff≥R. When considering experimental data it is found that fluorescence polarization anisotropy (FPA), a technique very sensitive to spinning, also yields apparent DNA radii depending upon fragment length. In order to derive DNA parameters which are independent of fragment length, we introduce a procedure for fitting FPA data which takes into account thermal distortions and employs the parametric expressions for rigid body rotations, spinning and tumbling, depending only upon L, P, and the actual DNA radius, R. Then the apparent persistence length P can be estimated once a value of R is assumed together with the value of the dynamic persistence length, the latter affecting the internal bending motions of the fragments. Fitting the FPA data is easily accomplished with the value of R=10 Å as suggested by a number of recent measurements. © 1996 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Abstract The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data. [ABSTRACT FROM AUTHOR]

Abstract A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 met single strand sequence onto the complementary purinic sequence centred on a 27 met duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10-90 Celsius. The Tm of the triplex, 37 Celsius, was well separated from the onset of duplex melting. When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the Tm of the triplex was shifted to approximately 32 Celsius and overlapped the melting of the duplex. -We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and triplex samples to determine the hydrodynamic parameters in the temperature range 10-40 Celsius. The fluorescence lifetime of EB in the samples of double and triple stranded DNA is the same (21.3 +/- 0.5 ns) at 20 Celsius, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal triplex, and nicked triplex samples were 10.7 +/- 0.2, 12.2 +/- 0.2, and 12.0 +/- 0.2 Angstrom. FPA measurements on normal triplex DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the triplex carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal triplex sample. The torsional rigidity of the triplex forms was found to be higher than that of the duplex form. [ABSTRACT FROM AUTHOR]

Background: With the passing of Jörg Langowski 6 May 2017 in a sailplane accident, the scientific community was deprived of a strident and effective voice for DNA and chromatin molecular and computational biophysics, for open access publishing and for the creation of effective scientific research networks. Methods: Here, after reviewing some of Jörg's key research contributions and ideas, we offer through the personal remembrance of his closest collaborators, a deep analysis of the major results of his research and the future directions they have engendered. Conclusions: The legacy of Jörg Langowski has been to propel a way of viewing biological function that considers living systems as dynamic and in three dimensions. This physical view of biology that he pioneered is now, finally, becoming established also because of his great effort. [ABSTRACT FROM AUTHOR]