Phototoxicity induced in living HeLa cells by focused femtosecond laser pulses: a data-driven approach.

Nonlinear optical microscopy is a powerful label-free imaging technology, providing biochemical and structural information in living cells and tissues. A possible drawback is photodamage induced by high-power ultrashort laser pulses. Here we present an experimental study on thousands of HeLa cells, to characterize the damage induced by focused femtosecond near-infrared laser pulses as a function of laser power, scanning speed and exposure time, in both wide-field and point-scanning illumination configurations. Our data-driven approach offers an interpretation of the underlying damage mechanisms and provides a predictive model that estimates its probability and extension and a safety limit for the working conditions in nonlinear optical microscopy. In particular, we demonstrate that cells can withstand high temperatures for a short amount of time, while they die if exposed for longer times to mild temperatures. It is thus better to illuminate the samples with high irradiances: thanks to the nonlinear imaging mechanism, much stronger signals will be generated, enabling fast imaging and thus avoiding sample photodamage.

Hyperspectral imaging with a TWINS birefringent interferometer.

We introduce a high-performance hyperspectral camera based on the Fourier-transform approach, where the two delayed images are generated by the Translating-Wedge-Based Identical Pulses eNcoding System (TWINS) [Opt. Lett. 37, 3027 (2012)], a common-path birefringent interferometer that combines compactness, intrinsic interferometric delay precision, long-term stability and insensitivity to vibrations. In our imaging system, TWINS is employed as a time-scanning interferometer and generates high-contrast interferograms at the single-pixel level. The camera exhibits high throughput and provides hyperspectral images with spectral background level of -30dB and resolution of 3 THz in the visible spectral range. We show high-quality spectral measurements of absolute reflectance, fluorescence and transmission of artistic objects with various lateral sizes.

Implementation of an Automatic Stop Order and Initial Antibiotic Exposure in Very Low Birth Weight Infants.

Objective To evaluate if an antibiotic automatic stop order (ASO) changed early antibiotic exposure (use in the first 7 days of life) or clinical outcomes in very low birth weight (VLBW) infants. Study Design We compared birth characteristics, early antibiotic exposure, morbidity, and mortality data in VLBW infants (with birth weight <= 1500 g) born 2 years before (pre-ASO group, n = 313) to infants born in the 2 years after (post-ASO, n = 361) implementation of an ASO guideline. Early antibiotic exposure was quantified by days of therapy (DOT) and antibiotic use > 48 hours. Secondary outcomes included mortality, early mortality, early onset sepsis (EOS), and necrotizing enterocolitis. Results Birth characteristics were similar between the two groups. We observed reduced median antibiotic exposure (pre-ASO: 6.5 DOT vs. Post-ASO: 4 DOT; p < 0.001), and a lower percentage of infants with antibiotic use > 48 hours (63.4 vs. 41.3%; p < 0.001). There were no differences in mortality (12.1 vs 10.2%; p = 0.44), early mortality, or other reported morbidities. EOS accounted for less than 10% of early antibiotic use. Conclusion Early antibiotic exposure was reduced after the implementation of an ASO without changes in observed outcomes.

Size and nanocrystallinity controlled gold nanocrystals: synthesis, electronic and mechanical properties.

The influence of nanocrystallinity on the electronic and mechanical properties of metal nanoparticles is still poorly understood, due to the difficulty in synthesizing nanoparticles with a controlled internal structure. Here, we report on a new method for the selective synthesis of Au nanoparticles in either a single-domain or a polycrystalline phase maintaining the same chemical environment. We obtain quasi-spherical nanoparticles whose diameter is tunable from 6 to 13 nm with a resolution down to ≈0.5 nm and narrow size distribution (4-5%). The availability of such high-quality samples allows the study of the impact of the particle size and nanocrystallinity on a number of parameters, such as plasmon dephasing time, electron-phonon coupling, period and damping time of the radial breathing modes.

Tracking the primary photoconversion events in rhodopsins by ultrafast optical spectroscopy.

Opsins are a broad class of photoactive proteins, found in all classes of living beings from bacteria to higher animals, which work either as light-driven ion pumps or as visual pigments. The photoactive function in opsins is triggered by the ultrafast isomerization of the retinal chromophore around a specific carbon double bond, leading to a highly distorted, spectrally red-shifted photoproduct. Understanding, by either experimental or computational methods, the time course of this photoisomerization process is of utmost importance, both for its biological significance and because opsin proteins are the blueprint for molecular photoswitches. This paper focuses on the ultrafast 11-cis to all-trans isomerization in visual rhodopsins, and has a twofold goal: (i) to review the most recent experimental and computational efforts aimed at exposing the very early phases of photoconversion; and (ii) discuss future advanced experiments and calculations that will allow an even deeper understanding of the process. We present high time resolution pump-probe data, enabling us to follow the wavepacket motion through the conical intersection connecting excited and ground states, as well as femtosecond stimulated Raman scattering data allowing us to track the subsequent structural evolution until the first stable all-trans photoproduct is reached. We conclude by introducing computational results for two-dimensional electronic spectroscopy, which has the potential to provide even greater detail on the evolution of the electronic structure of retinal during the photoisomerization process.

Ultrafast energy transfer and excited state coupling in an artificial photosynthetic antenna.

We have studied the energy transfer dynamics in an artificial light-harvesting dyad composed of a phthalocyanine (Pc) covalently linked to a carotenoid (Car). The combination of high temporal resolution transient absorption spectroscopy with global and target analysis allowed us to quantify the efficiency of the energy transfer from the S2 excited state of the Car to the Pc at 37%, close to values observed in some natural light-harvesting complexes. In addition, following selective excitation of the Pc, we have identified the spectral signatures of the S1 excited state of the Car which appear within the ≈30 fs time resolution of our measurement. This strongly indicates excited state coupling between the S1 state of Car and the Qx state of Pc, with important implications for the regulation of photosynthetic activity.

Explaining the temperature dependence of spirilloxanthin's S* signal by an inhomogeneous ground state model.

We investigate the nature of the S* excited state in carotenoids by performing a series of pump-probe experiments with sub-20 fs time resolution on spirilloxanthin in a polymethyl-methacrylate matrix varying the sample temperature. Following photoexcitation, we observe sub-200 fs internal conversion of the bright S2 state into the lower-lying S1 and S* states, which in turn relax to the ground state on a picosecond time scale. Upon cooling down the sample to 77 K, we observe a systematic decrease of the S*/S1 ratio. This result can be explained by assuming two thermally populated ground state isomers. The higher lying one generates the S* state, which can then be effectively frozen out by cooling. These findings are supported by quantum chemical modeling and provide strong evidence for the existence and importance of ground state isomers in the photophysics of carotenoids.

Broadband optical near-field microscope for nanoscale absorption spectroscopy of organic materials.

Conjugated organic materials in the solid state are generally amorphous or polycrystalline, with local order only achieved in mesoscopic domains with size ranging from a few tens to a few hundreds of nanometres. Understanding the interplay between mesoscopic order and macroscopic behaviour of these materials calls for a spatially resolved study of their optical properties. Near-field scanning optical microscopy allows one in principle to beat the diffraction limit in optical imaging. A quantitative measurement of nanoscale absorption spectra is, however, complicated by the difficulty of obtaining broadband near-field illumination with sufficiently high intensity. Here we demonstrate a near-field spectrometer with 100-nm spatial resolution based on an ultrabroadband Ti : sapphire oscillator coupled to an aperture-based near-field scanning optical microscopy, enabling structural phase-selective imaging of organic materials at the nanoscale. In polycrystalline phtalocyanine films we can distinguish between the crystalline and the amorphous phase, thus providing previously unavailable information on their mesoscopic texture.

Mapping local field enhancements at nanostructured metal surfaces by second-harmonic generation induced in the near field.

We report on an aperture scanning near-field optical microscope in which femtosecond pulses are coupled to a hollow-pyramid aperture sensor. Such probe displays high throughput and preserves pulse duration and polarization, enabling the achievement of sufficiently high peak power in the near field to perform nonlinear optics on the nanoscale. We use the system to observe the nonlinear optical response of nanostructured metal surfaces with sub-100-nm spatial resolution. We study second-harmonic generation from gold nanoparticles both isolated and in high-density patterns, highlighting a strong dependence of the generation efficiency on the shape and on the fine structure of the nanoemitter. In particular, we present results on closely packed gold triangles as well as on nanoellipsoids with different local surface plasmon resonances.

Coherent orbital waves in the photo-induced insulator-metal dynamics of a magnetoresistive manganite.

Photo-excitation can drive strongly correlated electron insulators into competing conducting phases, resulting in giant and ultrafast changes of their electronic and magnetic properties. The underlying non-equilibrium dynamics involve many degrees of freedom at once, whereby sufficiently short optical pulses can trigger the corresponding collective modes of the solid along temporally coherent pathways. The characteristic frequencies of these modes range between the few GHz of acoustic vibrations to the tens or even hundreds of THz for purely electronic excitations. Virtually all experiments so far have used 100 fs or longer pulses, detecting only comparatively slow lattice dynamics. Here, we use sub-10-fs optical pulses to study the photo-induced insulator-metal transition in the magnetoresistive manganite Pr(0.7)Ca(0.3)MnO(3). At room temperature, we find that the time-dependent pathway towards the metallic phase is accompanied by coherent 31 THz oscillations of the optical reflectivity, significantly faster than all lattice vibrations. These high-frequency oscillations are suggestive of coherent orbital waves, crystal-field excitations triggered here by impulsive stimulated Raman scattering. Orbital waves are likely to be initially localized to the small polarons of this room-temperature manganite, coupling to other degrees of freedom at longer times, as photo-domains coalesce into a metallic phase.

Conjugation length dependence of internal conversion in carotenoids: role of the intermediate state.

We report on a sub-20-fs transient absorption study of the S2(1(1)B(+)(u))-->S1(2(1)A(-)(g)) internal conversion in a series of carotenoids with a number of conjugated double bonds (N) ranging from 5 to 15. For the longer carotenoids (N>or=9), the measurements reveal the existence of an additional intermediate excited state lying between the optically allowed S2 state and the lower-lying forbidden S1 state. This state enables us to explain the nonmonotonic dependence of the S2-->S1 conversion rate on N and is expected to play an important role in photosynthetic light harvesting.

Highly efficient second-harmonic nanosource for near-field optics and microscopy.

A nanometric source of second-harmonic (SH) light with unprecedented efficiency is demonstrated; it exploits the grazing-incidence illumination of a metal tip, which is conventionally used for atomic force microscopy, by 25-fs laser pulses of a high-energy Ti:sapphire oscillator. Tip scanning around the beam focus shows that the SH generation is strongly localized at its apex. The polarization dependence of the SH light complies with the model of an on-axis nonlinear oscillating dipole.

Photosynthetic light harvesting by carotenoids: detection of an intermediate excited state.

We present the first direct evidence of the presence of an intermediate singlet excited state (Sx) mediating the internal conversion from S2 to S1 in carotenoids. The S2 to Sx transition is extremely fast and is completed within approximately 50 femtoseconds. These results require a reassessment of the energy transfer pathways from carotenoids to chlorophylls in the primary step of photosynthesis.

Femtosecond micromachining of symmetric waveguides at 1.5 microm by astigmatic beam focusing.

We report on a new spatial beam-shaping approach for fabrication of waveguides with a circular transverse profile by femtosecond laser pulses, using an astigmatic beam and controlling both beam waist and focal position in the tangential and sagittal planes. We apply this technique to write single-mode active waveguides at 1.5microm in Er:Yb-doped glass substrates. The experimental results are well described by a simple nonlinear absorption model.

Measurement of rectal blood flow by laser Doppler flowmetry in inflammatory bowel disease.

BACKGROUND/AIMS: Vascular alterations have been suggested as pathogenic factors in inflammatory bowel disease, particularly Crohn's disease. The aim of our study was to assess rectal blood flow in patients with active inflammatory bowel disease involving the rectum. METHODOLOGY: Endoscopic measurement of rectal blood flow was performed by laser Doppler flowmetry in 45 subjects divided into three groups: healthy controls, ulcerative colitis and rectal Crohn's disease. RESULTS: Rectal perfusion was found to be significantly impaired in patients with ulcerative colitis, but not in those with Crohn's colitis. CONCLUSIONS: Our results confirm the role of local ischemia in ulcerative colitis, but do not support the theory that vascular factors play a key role in the pathogenesis of Crohn's disease.

Rectal blood flow in ulcerative colitis.

OBJECTIVE: To determine the state of local blood flow in ulcerative colitis. METHODS: Rectal blood flow was measured by means of laser Doppler flowmetry during endoscopy in 20 patients with active or inactive ulcerative colitis and in 20 healthy controls. RESULTS: A significant (p < 0.01) reduction in the values of rectal perfusion was observed in ulcerative colitis patients both in active phase and in clinical and endoscopic remission. CONCLUSIONS: Impaired local blood flow may have a pathogenetic role in ulcerative colitis. Remission of the disease is not accompanied by normalization of local microcirculation, which may predispose to relapses.