Molecular understanding of label-free second harmonic imaging of microtubules.

Microtubules are a vital component of the cell's cytoskeleton and their organization is crucial for healthy cell functioning. The use of label-free SH imaging of microtubules remains limited, as sensitive detection is required and the true molecular origin and main determinants required to generate SH from microtubules are not fully understood. Using advanced correlative imaging techniques, we identified the determinants of the microtubule-dependent SH signal. Microtubule polarity, number and organization determine SH signal intensity in biological samples. At the molecular level, we show that the GTP-bound tubulin dimer conformation is fundamental for microtubules to generate detectable SH signals. We show that SH imaging can be used to study the effects of microtubule-targeting drugs and proteins and to detect changes in tubulin conformations during neuronal maturation. Our data provide a means to interpret and use SH imaging to monitor changes in the microtubule network in a label-free manner.

Photoluminescence as a Probe of the Electrical Charge Dependence of Gold Nanoparticles.

Electro-optical switching can be achieved by changing the optical absorption of metal nanoparticles by adding or removing electrical charge, corresponding to increased, respectively, decreased electron density. In this work a different approach is taken by changing the photoluminescence properties as a function of electrical charge on gold nanoparticles. Whereas larger gold nanoparticles (diameter d = 5 and 10 nm), exhibiting a plasmon resonance peak in the absorption spectrum, were used to measure changes of the optical absorption spectrum upon electrical charging, for smaller gold nanoparticles (d = 2 and 5 nm) electrical charging was observed via changes of the photoluminescence. Increase and decrease in photoluminescence was observed at positive and negative applied potentials, respectively. The relation between changes of optical absorption and photoluminescence for the 5 nm particles by electrical charging provides information on the influence of the charge state on the electronic properties and therefore the optical transition probability. The reported observation that not only the optical absorption, but also the photoluminescence is affected by alteration of the electrical charge onto gold nanoparticles may open a new way towards electro-optical switching and bio-sensing.

Controlling the photoluminescence of CdSe/ZnS quantum dots with a magnetic field.

We present an investigation of the photoluminescence of CdSe/ZnS quantum dots at high light intensity and in low magnetic fields. Upon increasing the magnetic field up to 90 G, the photoluminescence intensity drops. When decreasing the magnetic field back to zero the photoluminescence drop remains present. A plausible explanation is the Zeeman splitting of defect-associated energy levels under the influence of a magnetic field. The defect-trapped electrons may then be positioned at a metastable level, thereby reducing the number of recombinations. This effect may be used to control the luminescence of quantum dots.

A comparison of the pentaammine(pyridyl)ruthenium(II) and 4-(dimethylamino)phenyl groups as electron donors for quadratic non-linear optics.

Hyper-Rayleigh scattering and Stark spectroscopic studies show that the complex salts [1-4]PF6 have larger static first hyperpolarizabilities beta 0 than [5-8]PF6, because the higher HOMO energy of a (RuII(NH3)5)2+ centre more than offsets the superior pi-orbital overlap in the purely organic chromophores.

Theoretical upper limits and experimental overestimates for molecular hyperpolarizabilities: a symbiosis.

A recently developed theory concerning the physical limits on optical hyperpolarizabilities is shown to be useful in the theoretical prediction of contributions of multiphoton fluorescence to experimentally determined hyperpolarizability values. Recent experimental results are compared with the theoretical limit. Without correction for the fluorescence contribution, results for the first hyperpolarizability in the forbidden region can be obtained. Proper correction of the systematic error shifts these values to the theoretical limit. Hence, experimental values above the theoretical upper limit should be suspect, but an appropriate fluorescence suppression technique is available. Reversing the argument, the observation that artificially overestimated values for the first hyperpolarizability are reduced from the forbidden region to exactly this upper limit is found to corroborate the limiting theory.

Bacteriorhodopsin: a natural (nonlinear) photonic bandgap material.

The angular dependence of the hyper-Rayleigh scattered light intensity from a suspension of bacteriorhodopsin has been analyzed. The observation calls for combination of the second-order nonlinear hyperpolarizability of the retinal chromophore with the linear refractive index of the apo-protein matrix. The structuring of the small nonlinear chromophore in the large linear matrix imparts properties of a photonic bandgap to bacteriorhodopsin.

High-frequency demodulation of multiphoton fluorescence in long-wavelength hyper-Rayleigh scattering.

Suppression of the multiphoton fluorescence contribution to the hyper-Rayleigh (second-order nonlinear optical) scattering signal was recently achieved by intrinsic demodulation of the fluorescence at high amplitude-modulation (AM) frequencies [Olbrechts et al., Rev. Sci. Instrum. 69, 2233 (1998)]. These high AM frequencies were obtained from the high harmonic content in the Fourier spectrum of a repetitive train of femtosecond pulses from a Ti:sapphire laser emitting at 800 nm. We have used a femtosecond parametric oscillator to shift the fundamental wavelength to 1.3 mum . By further improving the detection electronics, we can now obtain fluorescence suppression at AM frequencies up to 600 MHz. Fluorescence-free hyperpolarizability values were obtained for fluorescent dipolar compounds as well as for an ionic fluorophore. The results also indicate that shifting the fundamental wavelength to the near infrared only is not a general solution to the multiphoton fluorescence problem in hyper-Rayleigh scattering.

Nonlinear optical properties of proteins measured by hyper-rayleigh scattering in solution.

Hyper-Rayleigh scattering has been used to determine the nonlinear optical properties of a chromophore-containing protein in solution. Because the technique of hyper-Rayleigh scattering allows the measurement of hyperpolarizabilities in an isotropic solution without the application of an electric field, this method is ideally suited for the study of proteins that carry a net charge. The observed orientational correlation between the nonlinear optical chromophores in incompletely solubilized protein molecules suggests that guidelines from protein structures can be used for the engineering of supramolecular structures with high optical nonlinearity.

Determination of the hyperpolarizability of an octopolar molecular ion by hyper-Rayleigh scattering.

The first observation to our knowledge of a second-order nonlinear optical effect in a nonpolar molecular ion is reported. Hyper-Rayleigh scattering was observed from aqueous and organic solutions of the tricyanomethanide ion [C(CN)(3)](-). Because of the absence of a dipole moment and the presence of a charge in this planar, nonpolar, but highly polarizable molecular anion with D(3h) symmetry, no electric-field-induced second-harmonic-generation experiments are possible. The experimental values for the first hyperpolarizability beta(xxx) [(7 +/- 1.5) x 10(-30) esu, independent of the solvent] are in good agreement with the results from quantum-chemical calculations.

A fluorescence lifetime study of virginiamycin S using multifrequency phase fluorometry.

Using multifrequency phase fluorometry, fluorescence lifetimes have been assigned to the different protolytic forms of the antibiotic virginiamycin S. These lifetimes are 0.476 +/- 0.005 ns for the uncharged form, 1.28 +/- 0.2 and 7.4 +/- 0.2 ns for the zwitterionic form, 1.19 +/- 0.01 ns for the negatively charged form, and 1.9 +/- 0.1 ns for the double negatively charged form. The assignments are based on lifetime measurements as a function of pH, volume percent ethanol, and excitation wavelength. Excited-state proton transfer is taken into account. It is complete at pH values lower than 1, and no fluorescence of the fully protonated charged form is observed. At pH 8, an excited-state pK* increase is calculated, but proton association is too slow to cause excited-state proton transfer. The addition of divalent cations, at pH 9.4, increases the lifetime of the negatively charged form to a value dependent upon the specific nature of the cation (7.58 +/- 0.06 ns for Mg2+, 6.54 +/- 0.02 ns for Ca2+, and 3.74 +/- 0.05 ns for Ba2+). Monovalent cations do not influence the lifetimes, indicating that their binding to the macrocycle does not influence the fluorescent moiety. The model compound 3-hydroxypicolinamide shows an analogous behavior, but the retrieved lifetime can differ significantly.

Interaction between virginiamycin S and ribosomes is partly provided by a salt bridge with a Mg2+ ion.

Type B streptogramins, such as virginiamycin S (VS), are cyclic hexadepsipeptides, inhibiting protein synthesis in prokaryotes. L-Thr connects a 3-hydroxypicolinyl residue (3-OH-Pic) to the peptide lactone ring. The fluorescence intensity of 3-OH-Pic is strongly increased by chelation to alkaline earth cations or binding to ribosomes. Similar behavior of the ribosome-VS complex and the VS-Mg chelate provides strong evidence for the presence of a VS-Mg chelate within the ribosomal binding site. Different models involving the ribosome binding of either members of the VS-Mg2+ chelate or both have been tested by fluorescence lifetime measurements, equilibrium titrations, and stopped-flow spectrofluorometry. Our data strongly suggest that (a) the interaction between VS and the ribosome is partly provided by a salt bridge between suitable acceptor atoms of the ribosome and the 3-OH-Pic residue, (b) Mg2+ can be exchanged by Mn2+ without dissociation of the ribosome-VS complex, (c) Mg2+ coordinates to the negative form of the 3-OH-Pic residue, probably via an interaction with the phenolate oxygen and the amide carboxyl group, and (d) the picolinyl residue is essential for the biological activity, as indicated by the lack of activity when the latter is replaced by a serine derivative.

Large-bowel perforation. A rare complication of intravesical Nd-YAG laser irradiation of bladder tumors.

A case report of large-bowel perforation after intravesical neodymium-yttrium aluminum garnet laser irradiation is presented. Recommendations to prevent bowel injury are discussed.

Reduction of phase instability in a mode-locked argon laser.

Timing fluctuations in a mode-locked laser cause phase noise sidebands on the carrier and its harmonics in the frequency spectrum of the output of the laser. We reduced the phase noise of the output of a mode-locked argon laser by 40 dB, 80 Hz away from the carrier, as measured on its twentieth harmonic by simply replacing the commonly supplied rf synthesizer by a higher performance frequency generator. Applications demanding reduced timing fluctuations or reduced phase noise include synchronization of lasers and cross-correlation detection as in phase fluorometry.