Multi-derivative method for phase extraction without knowing carrier frequencies in off-axis quantitative phase imaging.

We propose a multi-derivative method to reconstruct the phase of transparent objects in off-axis quantitative phase imaging (QPI). By numerically computing first-, second-, and third-order derivatives of the interferogram, we demonstrate that one can extract the quantitative phase information in a straightforward way, without prior knowledge of the carrier frequencies or Fourier transform. In contrast to existing advanced derivative methods, our approach markedly streamlines the alignment and retrieval processes, all without requiring any special prerequisites. This enhancement seamlessly translates into improved reconstruction quality. Furthermore, when compared to cutting-edge Fourier-division-based methods, our technique distinctly accelerates the phase retrieval speed. We verified our method using white-light diffraction phase microscopy and laser off-axis QPI, and the results indicate that our method can allow a fast, high-quality retrieval with frame rates up to 41.6 fps for one- megapixel interferograms on a regular computer.

Laboratory evidence of magnetic reconnection hampered in obliquely interacting flux tubes.

Magnetic reconnection can occur when two plasmas, having anti-parallel components of the magnetic field, encounter each other. In the reconnection plane, the anti-parallel component of the field is annihilated and its energy released in the plasma. Here, we investigate through laboratory experiments the reconnection between two flux tubes that are not strictly anti-parallel. Compression of the anti-parallel component of the magnetic field is observed, as well as a decrease of the reconnection efficiency. Concomitantly, we observe delayed plasma heating and enhanced particle acceleration. Three-dimensional hybrid simulations support these observations and highlight the plasma heating inhibition and reconnection efficiency reduction for these obliquely oriented flux tubes.

Anti-staphylococcal activity and mode of action of thioridazine photoproducts.

Antibiotic resistance became an increasing risk for population health threatening our ability to fight infectious diseases. The objective of this study was to evaluate the activity of laser irradiated thioridazine (TZ) against clinically-relevant bacteria in view to fight antibiotic resistance. TZ in ultrapure water solutions was irradiated (1-240 min) with 266 nm pulsed laser radiation. Irradiated solutions were characterized by UV-Vis and FTIR absorption spectroscopy, thin layer chromatography, laser-induced fluorescence, and dynamic surface tension measurements. Molecular docking studies were made to evaluate the molecular mechanisms of photoproducts action against Staphylococcus aureus and MRSA. More general, solutions were evaluated for their antimicrobial and efflux inhibitory activity against a panel of bacteria of clinical relevance. We observed an enhanced antimicrobial activity of TZ photoproducts against Gram-positive bacteria. This was higher than ciprofloxacin effects for methicillin- and ciprofloxacin-resistant Staphylococcus aureus. Molecular docking showed the Penicillin-binding proteins PBP3 and PBP2a inhibition by sulforidazine as a possible mechanism of action against Staphylococcus aureus and MRSA strains, respectively. Irradiated TZ reveals possible advantages in the treatment of infectious diseases produced by antibiotic-resistant Gram-positive bacteria. TZ repurposing and its photoproducts, obtained by laser irradiation, show accelerated and low-costs of development if compared to chemical synthesis.

Spectroscopic Characterization of Emulsions Generated with a New Laser-Assisted Device.

This paper presents a spectroscopic study of emulsions generated with a laser-assisted device. Fourier transform infrared (FTIR), Raman and UV-Vis-NIR reflectance spectra of emulsions, recorded before and after exposure to laser radiation were used to characterize the effect of laser irradiation. The paper also presents a comparison between the calculated IR spectra and the experimental FTIR spectra of an emulsion's components. FTIR measurements allowed the identification of absorption bands specific to each of the emulsions' components. Moreover, it enabled the observation of destabilization of the emulsion in real-time. Raman spectroscopy allowed the observation of the modifications at a molecular level, by identifying the vibrations of the representative functional groups and the polymerization of sodium tetradecyl sulfate (STS) molecules by analyzing the evolution of the carbonyl band. UV-Vis-NIR reflectance spectra of emulsions before and after exposure to laser radiation showed that the physical characteristics of the emulsions changed during irradiation-the dimensions of the droplets decreased, leading to an emulsion with a better time stability. These results proved that the employed spectroscopy techniques were powerful tools in emulsion analysis.

Laser-driven radiation: Biomarkers for molecular imaging of high dose-rate effects.

Recently developed short-pulsed laser sources garner high dose-rate beams such as energetic ions and electrons, x rays, and gamma rays. The biological effects of laser-generated ion beams observed in recent studies are different from those triggered by radiation generated using classical accelerators or sources, and this difference can be used to develop new strategies for cancer radiotherapy. High-power lasers can now deliver particles in doses of up to several Gy within nanoseconds. The fast interaction of laser-generated particles with cells alters cell viability via distinct molecular pathways compared to traditional, prolonged radiation exposure. The emerging consensus of recent literature is that the differences are due to the timescales on which reactive molecules are generated and persist, in various forms. Suitable molecular markers have to be adopted to monitor radiation effects, addressing relevant endogenous molecules that are accessible for investigation by noninvasive procedures and enable translation to clinical imaging. High sensitivity has to be attained for imaging molecular biomarkers in cells and in vivo to follow radiation-induced functional changes. Signal-enhanced MRI biomarkers enriched with stable magnetic nuclear isotopes can be used to monitor radiation effects, as demonstrated recently by the use of dynamic nuclear polarization (DNP) for biomolecular observations in vivo. In this context, nanoparticles can also be used as radiation enhancers or biomarker carriers. The radiobiology-relevant features of high dose-rate secondary radiation generated using high-power lasers and the importance of noninvasive biomarkers for real-time monitoring the biological effects of radiation early on during radiation pulse sequences are discussed.

In vitro antimicrobial efficacy of laser exposed chlorpromazine against Gram-positive bacteria in planktonic and biofilm growth state.

Aqueous chlorpromazine solutions exposed to 266 nm generated as fourth harmonic of Nd:YAG pulsed laser along time intervals from 1 min to 240 min were investigated for their antimicrobial activity against planktonic and adherent Gram-positive bacterial strains. Qualitative and quantitative assays based on microbiological methods and flow cytometry assays were performed to establish the minimum inhibitory and minimum biofilm eradication concentrations and to reveal some of the possible mechanisms of antimicrobial activity. Optimal irradiation conditions and combinations of photoproducts for achieving the best antimicrobial and antibiofilm effects are suggested. It was confirmed that chlorpromazine solutions irradiated for 15 min and 30 min have the best antimicrobial and antibiofilm activity against Staphylococcus aureus ATCC 6538, methicillin susceptible Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Enterococcus faecium 17-VAR, Enterococcus faecalis 2921, and Bacillus subtilis 6633. Flow cytometry revealed that two of the possible mechanisms of the antimicrobial activity of irradiated chlorpromazine are the inhibition of efflux pumps activity and induction of cellular membrane lesions.

Optical excitation and detection of neuronal activity.

Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can modulate behavior in live organisms. However, detecting the response to the optical stimulation requires electrophysiology with physical contact or fluorescent imaging at target locations, which is often limited by photobleaching and phototoxicity. In this paper, we show that phase imaging can report the intracellular transport induced by optogenetic stimulation. We developed a multimodal instrument that can both stimulate cells with subcellular spatial resolution and detect optical pathlength (OPL) changes with nanometer scale sensitivity. We found that OPL fluctuations following stimulation are consistent with active organelle transport. Furthermore, the results indicate a broadening in the transport velocity distribution, which is significantly higher in stimulated cells compared to optogenetically inactive cells. It is likely that this label-free, contactless measurement of optogenetic response will provide an enabling approach to neuroscience.

Hyperpolarised NMR to follow water proton transport through membrane channels via exchange with biomolecules.

Water uptake in vesicles and the subsequent exchange between water protons and amide -NH protons in amino acids can be followed by a new, highly sensitive, type of magnetic resonance spectroscopy: dynamic nuclear polarisation (DNP)-enhanced NMR in the liquid state. Water hydrogen atoms are detected prior to and after their transfer to molecular sites in peptides and proteins featuring highly-accessible proton-exchangeable groups, as is the case for the -NH groups of intrinsically disordered proteins. The detected rates for amide proton-water proton exchange can be modulated by membrane-crossing rates, when a membrane channel is interposed. We hyperpolarised water proton spins via dynamic nuclear polarisation followed by sample dissolution (d-DNP) and transferred the created polarisation to -NH groups with high solvent accessibility in an intrinsically disordered protein domain. This domain is the membrane anchor of c-Src kinase, whose activity controls cell proliferation. The hindrance of effective water proton transfer rate constants observed in free solvent when a membrane-crossing step is involved is discussed. This study aims to assess the feasibility of recently-introduced hyperpolarised (DNP-enhanced) NMR to assess water membrane crossing dynamics.

White-light diffraction phase microscopy at doubled space-bandwidth product.

White light diffraction microscopy (wDPM) is a quantitative phase imaging method that benefits from both temporal and spatial phase sensitivity, granted, respectively, by the common-path geometry and white light illumination. However, like all off-axis quantitative phase imaging methods, wDPM is characterized by a reduced space-bandwidth product compared to phase shifting approaches. This happens essentially because the ultimate resolution of the image is governed by the period of the interferogram and not just the diffraction limit. As a result, off-axis techniques generates single-shot, i.e., high time-bandwidth, phase measurements, at the expense of either spatial resolution or field of view. Here, we show that combining phase-shifting and off-axis, the original space-bandwidth is preserved. Specifically, we developed phase-shifting diffraction phase microscopy with white light, in which we measure and combine two phase shifted interferograms. Due to the white light illumination, the phase images are characterized by low spatial noise, i.e., <1nm pathlength. We illustrate the operation of the instrument with test samples, blood cells, and unlabeled prostate tissue biopsy.

Statistical dispersion relation for spatially broadband fields.

The dispersion relation is fundamental to a physical phenomenon that develops in both space and time. This equation connects the spatial and temporal frequencies involved in the dynamic process through the material constants. Electromagnetic plane waves propagating in homogeneous media are bound by simple dispersion relation, which sets the magnitude of the spatial frequency, k, as being proportional to the temporal frequency, ω, with the proportionality constant dependent on the refractive index, n, and the speed of light in vacuum, c. Here we show that, for spatially broadband fields, an analog dispersion relation can be derived, except in this case the k-vector variance is connected with the temporal frequency through the statistics of the refractive index fluctuations in the medium. We discuss how this relationship can be used to retrieve information about refractive index distributions in biological tissues. This result is particularly significant in measurements of angular light scattering and quantitative phase imaging of biological structures.

Chlorpromazine transformation by exposure to ultraviolet laser beams in droplet and bulk.

Multiple drug resistance requires a flexible approach to find medicines able to overcome it. One method could be the exposure of existing medicines to ultraviolet laser beams to generate photoproducts that are efficient against bacteria and/or malignant tumors. This can be done in droplets or bulk volumes. In the present work are reported results about the interaction of 266nm and 355nm pulsed laser radiation with microdroplets and bulk containing solutions of 10mg/ml Chlorpromazine Hydrochloride (CPZ) in ultrapure water. The irradiation effects on CPZ solution at larger time intervals (more than 30min) are similar in terms of generated photoproducts if the two ultraviolet wavelengths are utilized. The understanding of the CPZ parent compound transformation may be better evidenced, as shown in this paper, if studies at shorter than 30minute exposure times are made coupled with properly chosen volumes to irradiate. We show that at exposure to a 355nm laser beam faster molecular modifications of CPZ in ultrapure water solution are produced than at irradiation with 266nm, for both microdroplet and bulk volume samples. These effects are evidenced by thin layer chromatography technique and laser induced fluorescence measurements.

Characterization of mixtures of compounds produced in chlorpromazine aqueous solutions by ultraviolet laser irradiation: their applications in antimicrobial assays.

The study reports an investigation of the photoproducts obtained by exposure of chlorpromazine hydrochloride in ultrapure water (concentration 2 mg/mL) to a 266-nm laser beam obtained by fourth harmonic generation from a Nd:YAG laser (6-ns full time width at half maximum, 10-Hz pulse repetition rate). The photoproducts were analyzed by steady-state UV-Vis absorption, laser-induced fluorescence, Fourier transform infrared spectroscopy, and liquid chromatography-tandem time-of-flight mass spectroscopy. Two figures showing pathways that take place during irradiation for obtaining the final products are shown. The quantum yield of singlet oxygen generation by chlorpromazine (CPZ) was determined relative to standard Zn-phthalocyanine in dimethyl sulfoxide. To outline the role of fluorescence in photoproducts formation rates, fluorescence quantum yield of CPZ during exposure to 355-nm radiation (third harmonic of the fundamental beam of Nd:YAG laser) was investigated relative to standard Coumarin 1 in ethanol. The CPZ solutions exposed 60 and 240 min to 266-nm laser beam, respectively, were tested against Staphylococcus aureus ATCC 25923 strain. For 25 µL of CPZ samples irradiated 240 min, a higher diameter of inhibition has obtained against the tested strain than for the 60-min exposed ones.

The in vitro activity of products formed from exposure of chlorpromazine to a 266 nm laser beam against species of mycobacteria of human interest.

Chlorpromazine (CPZ) was exposed to a 266 nm laser beam for different periods of time ranging from minutes to 24 h. At intervals, the products from irradiation were evaluated by thin-layer chromatography (TLC) and evaluated for their activity against mycobacteria of human interest (Mycobacterium tuberculosis, M. avium, M. intracellulare and their corresponding reference strains or clinical isolates). With the exception of the M. avium 47/07 clinical strain, the products produced from the irradiation of CPZ for 4 h had greater activity against M. intracellulare ATCC, M. avium ATCC, H37Rv and the Multidrug-resistant tuberculosis (MDR-TB) strains as opposed to that produced by the unirradiated control. The level of products from the 4-h exposure of CPZ remained the same throughout the next 20 h of irradiation. Of significant note is that the irradiation products of CPZ had lower in vitro cytotoxicity against human cells, suggesting that this approach may be useful for the development of compounds more bioactive than the parental species.

Exposure of chlorpromazine to 266 nm laser beam generates new species with antibacterial properties: contributions to development of a new process for drug discovery.

INTRODUCTION: Phenothiazines when exposed to white light or to UV radiation undergo a variety of reactions that result in degradation of parental compound and formation of new species. This process is slow and may be sped up with exposure to high energy light such as that produced by a laser. METHODS: Varying concentrations of Chlorpromazine Hydrochloride (CPZ) (2-20 mg/mL in distilled water) were exposed to 266 nm laser beam (time intervals: 1-24 hrs). At distinct intervals the irradiation products were evaluated by spectrophotometry between 200-1500 nm, Thin Layer Chromatography, High Pressure Liquid Chromatography (HPLC)-Diode Array Detection, HPLC tandem mass spectrometry, and for activity against the CPZ sensitive test organism Staphylococcus aureus ATCC 25923. RESULTS: CPZ exposure to 266 nm laser beam of given energy levels yielded species, whose number increased with duration of exposure. Although the major species produced were Promazine (PZ), hydroxypromazine or PZ sulfoxide, and CPZ sulfoxide, over 200 compounds were generated with exposure of 20 mg/mL of CPZ for 24 hrs. Evaluation of the irradiation products indicated that the bioactivity against the test organism increased despite the total disappearance of CPZ, that is due, most probably, to one or more new species that remain yet unidentified. CONCLUSIONS: Exposure of CPZ to a high energy (6.5 mJ) 266 nm laser beam yields rapidly a large number of new and stable species. For biological grade phenothiazines (in other words knowing the impurities in the samples: solvent and solute) this process may be reproducible because one can control within reasonably low experimental errors: the concentration of the parent compound, the laser beam wavelength and average energy, as well as the duration of the exposure time. Because the process is "clean" and rapid, it may offer advantages over the pyrogenically based methods for the production of derivatives.

Direct modification of bioactive phenothiazines by exposure to laser radiation.

Whereas exposure of combinations of a phenothiazine and bacterium to incoherent UV increases the activity of the phenothiazine, exposure of the phenothiazine alone does not yield an increase of its activity. Because the laser beam energy is greater than that produced by the incoherent UV sources, exposure of phenothiazines to specific lasers may yield molecules with altered activities over that of the unexposed parent. Chlorpromazine, thioridazine and promethazine active against bacteria were exposed to two distinct lasers for varying periods of time. Absorption and fluorescence spectra were conducted prior to and post-exposure and the products of laser exposure evaluated for activity against a Staphylococcus aureus ATCC strain via a disk susceptibility assay. Exposure to lasers alters the absorption/fluorescence spectra of the phenothiazines; reduces the activity of thioridazine against the test bacterium; produces a highly active chlorpromazine compound against the test organism. Exposure of phenothiazines to lasers alters their structure that results in altered activity against a bacterium. This is the first report that lasers can alter the physico-chemico characteristics to the extent that altered bioactivity results. Exposure to lasers is expected to yield compounds that are difficult to make via chemical manipulation methods. A survey of selected patents of interest, even co-lateral for the subject of this article is shortly made.