Time-frequency characterization of femtosecond extreme ultraviolet pulses

A measurement of chirp and pulse duration of fifth harmonic of a frequency-doubled Ti:sapphire laser was presented. The photoelectron signal due to cross correlation of harmonics generated by 400 nm blue light and an 800 nm infrared probe pulse, was measured using energy resolved cross-correlation method. Results demonstrated that the technique could be used to characterize the time-frequency behavior of much higher-order harmonics

Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background
The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings
When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance
Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

Time-Frequency Characterization of Femtosecond Extreme Ultraviolet Pulses

A measurement of chirp and pulse duration of fifth harmonic of a frequency-doubled Ti:sapphire laser was presented. The photoelectron signal due to cross correlation of harmonics generated by 400 nm blue light and an 800 nm infrared probe pulse, was measured using energy resolved cross-correlation method. Results demonstrated that the technique could be used to characterize the time-frequency behavior of much higher-order harmonics

Two-color pump-probe experiments in helium using high-order harmonics

A pump-probe technique has been applied for measuring the lifetimes and absolute photoionization cross-sections of excited He states. The 1s2p 1P and 1s3p 1P states of He are excited by using the 13th and the 14th harmonic, respectively, of a tunable 70 ps dye laser generated in a Kr gas jet. The states are ionized after a varying time delay, by absorption of probe photons with energies between 1.6 and 4.5 eV. Lifetimes of $\tau(1s2p) = 0.57$ ns and $\tau(1s3p) = 1.76$ ns are determined with a precision of about 15%. A significant enhancement of the number of ions present in the lifetime curves at zero time delay for pressures above $6\times10^{-5}$ mbar is attributed to direct two-photon ionization of He in combination with AC Stark broadening of the excited state and absorption of the XUV light in the medium. Absolute photoionization cross-sections from the He 1s2p 1P and He 1s3p 1P states in the threshold region are determined by measuring the saturation of the ionization process with a precision of $\sim 25$%. In addition, the variation of the relative orientation between the polarization vectors of the pump and probe beams enables the determination of partial photoionization cross-sections

Time-frequency characterization of femtosecond extreme ultraviolet pulses.

We present energy-resolved cross-correlation measurements of an extreme ultraviolet (XUV) pulse, generated as the fifth harmonic (15.5 eV) of an intense 80 fs laser pulse centered at 400 nm. Spectrally resolving the cross-correlation signal allows us to characterize the time-dependent frequency of the XUV pulse. We find that the fifth harmonic has a small negative chirp in excess of that predicted by perturbation theory. In addition, by manipulating the chirp of the driving laser we can induce and measure a positive or a negative chirp on the XUV pulse

Osteopontin Reduces Biofilm Formation in a Multi-Species Model of Dental Biofilm

<div><p>Background</p><p>Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies <i>in vitro</i> model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms.</p><p>Methodology/Principal Findings</p><p>Biofilms consisting of <i>Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei</i> and <i>Streptococcus sanguinis</i> were grown in a flow cell system that permitted <i>in situ</i> microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence <i>in situ</i> hybridization, changed considerably in the presence of OPN. In particular, colonization of <i>S. mitis</i>, the best biofilm former in the model, was reduced dramatically.</p><p>Conclusions/Significance</p><p>OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed <i>in vivo</i>, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.</p></div

Biofilms grown in the presence of OPN, hybridized with EUB338 and species-specific probes SMIT, SSAN, ANAES, SDOW or SORA2.

<p>EUB338 targets all organisms in the biofilms and was labelled with Atto633 (red). Species-specific probes were labelled with Cy3 (green). <b>A.</b> <i>S. mitis</i> SK24, the dominant organism in biofilms grown without OPN, accounted for 14% of the bacterial biovolume. <b>B–F.</b> The relative biovolumes of all other organisms increased in biofilms grown with OPN, as compared to biofilms grown without OPN. <i>S. sanguinis</i> SK150 (<b>B</b>) was the most abundant organism in the biofilms (48% of the biovolume). <i>A. naeslundii</i> AK6 was a prominent colonizer in basal layers of the biofilms (<b>C</b>, 22% of the biovolume in the basal layer), but was detected less frequently in upper layers of the biofilm (<b>D</b>, 9% of the total biovolume). <i>S. downei</i> HG594 (<b>E</b>, 11% of the biovolume) and <i>S. oralis</i> SK248 (<b>F</b>, 3% of the biovolume) represented smaller fractions of the bacterial biofilm.</p

Bacterial composition of biofilms grown in the presence and absence of OPN.

<p>In biofilms grown without OPN (−OPN), <i>S. mitis</i> SK24 was the predominant organism. When OPN was present in the medium (+OPN), the abundance of <i>S. mitis</i> was dramatically lower, and the relative abundance of all other organisms increased. <i>S. sanguinis</i> SK150 became the predominant organism.</p