Survey of X-ray induced Cherenkov excited fluorophores with potential for human use.

X-ray induced molecular luminescence (XML) is a phenomenon that can be utilized for clinical, deep-tissue functional imaging of tailored molecular probes. In this study, a survey of common or clinically approved fluorophores was carried out for their megavoltage X-ray induced excitation and emission characteristics. We find that direct scintillation effects and Cherenkov generation are two possible ways to cause these molecules' excitation. To distinguish the contributions of each excitation mechanism, we exploited the dependency of Cherenkov radiation yield on X-ray energy. The probes were irradiated by constant dose of 6 MV and 18 MV X-ray radiation, and their relative emission intensities and spectra were quantified for each X-ray energy pair. From the ratios of XML, yield for 6 MV and 18 MV irradiation we found that the Cherenkov radiation dominated as an excitation mechanism, except for aluminum phthalocyanine, which exhibited substantial scintillation. The highest emission yields were detected from fluorescein, proflavin and aluminum phthalocyanine, in that order. XML yield was found to be affected by the emission quantum yield, overlap of the fluorescence excitation and Cherenkov emission spectra, scintillation yield. Considering all these factors and XML emission spectrum respective to tissue optical window, aluminum phthalocyanine offers the best XML yield for deep tissue use, while fluorescein and proflavine are most useful for subcutaneous or superficial use.

Assessment of the Content of Fluorescent Tracer in Granular Feed Mixture.

Background: This paper describes the use of fluorescence induced by UV radiation to evaluate the share of tracer in feed mixture. Methods: For the purpose of this study, three substances were used. They are as follows: Tinopal, Rhodamine B, and Uranine. Tracer in the form of maize or kardi was added to chicken feed before the mixing process. Grains used in the process were grinded in the mill sieve with a mesh size of 4 and 6 mm. The drawn samples of the mixture were illuminated with UV radiation to make grain tracer light, and then the photo was taken with a digital camera. The acquired images were analyzed with the use of a computer program running on the RGB color model, which was the way to obtain essential information about the percentage share of tracer. Results: It was observed that, in the case of kardi grains, the proposed method gives results significantly deviating from the verification method. Conclusions: Only the tests with the use of maize having an average particle diameter of 2.4 mm and tinted with the solution of Rhodamine B led to acceptable results (consensual with the predetermined verification level).

Retinocoroiditis asociada a tatuaje / Tattoo associated retinochoroiditis

CASO CLÍNICO: Una joven mujer fue referida por compromiso de la agudeza visual después de recibir un tercer tatuaje en su brazo. Se realizó evaluación sistémica y de laboratorio para excluir agentes infecciosos o enfermedad inflamatoria. Una lesión amarillenta yuxtafoveal junto con disrupción de la retina externa de tipo placoide y defectos pigmentarios focales fueron evaluados con imágenes multimodales. DISCUSIÓN: Los oftalmólogos que tratan uveítis deben considerar esta asociación poco común y preguntar a sus pacientes acerca de tatuajes e inflamación de estos, dado el número creciente de sujetos con tatuajes artísticos

CLINICAL CASE: A young woman was referred to our offices with impairment of visual acuity after she received a third tattoo on her arm. Systemic medical and laboratory work-up were performed in order to exclude an infectious agent or inflammatory disease. A yellowish juxtafoveal lesion in left eye along with a plaque-like outer retinal disruption and focal pigmentary defects was assessed using multi-modal diagnostic imaging. DISCUSSION: Ophthalmologists treating uveitis should consider this uncommon association and question patients regarding tattoos and tattoo inflammation given the rise of subjects undergoing artistic tattooing

Interaction and photodegradation characteristics of fluorescein dye in presence of ZnO nanoparticles.

The interaction between xanthene dye Fluorescein (Fl) and zinc oxide (ZnO) nanoparticles is investigated under physiological conditions. From the analysis of the steady state and time resolved spectroscopic studies in aqueous solution static mode is found to be responsible in the mechanism of fluorescence quenching of the dye Fl in presence of ZnO. ZnO nanoparticles are used as photocatalyst in order to degrade Fl dye. At pH 7, a maximum degradation efficiency of 44.4% of the dye has been achieved in presence of ZnO as a nanophotocatalyst and the photodegradation follows second-order kinetics.

Investigation of low-energy proton effects on aptamer performance for astrobiological applications.

Biochips are promising instruments for the search for organic molecules in planetary environments. Nucleic acid aptamers are powerful affinity receptors known for their high affinity and specificity, and therefore are of great interest for space biochip development. A wide variety of aptamers have already been selected toward targets of astrobiological interest (from amino acids to microorganisms). We present a first study to test the resistance of these receptors to the constraints of the space environment. The emphasis is on the effect of cosmic rays on the molecular recognition properties of DNA aptamers. Experiments on beam-line facilities have been conducted with 2 MeV protons and fluences much higher than expected for a typical mission to Mars. Our results show that this irradiation process did not affect the performances of DNA aptamers as molecular recognition tools.

Photoactive chitosan switching on bone-like apatite deposition.

The work was focused on the synthesis and characterization of the chitosan-g-fluorescein (CHFL) conjugate polymer as a biocompatible amphiphilic water-soluble photosensitizer, able to stimulate hydroxyapatite deposition upon visible light irradiation. Fluorescein (FL) grafting to chitosan (CH) chains was confirmed by UV-vis analysis of water solutions of FL and CHFL and by Fourier transform infrared spectroscopy (FTIR-ATR) analysis of CHFL and CH. Smooth CHFL cast films with 4 microm thickness were obtained by solvent casting. Continuous exposure to visible light for 7 days was found to activate the deposition of calcium phosphate crystals from a conventional simulated body fluid (SBF 1.0x) on the surface of CHFL cast films. EDX and FTIR-ATR analyses confirmed the apatite nature of the deposited calcium phosphate crystals. CHFL films preincubated in SBF (1.0x) solution under visible light irradiation and in the dark for 7 days were found to support the in vitro adhesion and proliferation of MG63 osteoblast-like cells (MTT viability test; 1-3 days culture time). On the other hand, the mineralization ability of MG63 osteoblast-like cells was significantly improved on CHFL films preincubated under visible light exposure (alkaline phosphatase activity (ALP) test for 1, 3, 7, and 14 days). The use of photoactive biocompatible conjugate polymer, such as CHFL, may lead to new therapeutic options in the field of bone/dental repair, exploiting the photoexcitation mechanism as a tool for biomineralization.

Investigation of neutron radiation effects on polyclonal antibodies (IgG) and fluorescein dye for astrobiological applications.

Detecting life in the Solar System is one of the great challenges of new upcoming space missions. Biochips have been proposed as a way to detect organic matter on extraterrestrial objects. A biochip is a miniaturized device composed of biologically sensitive systems, such as antibodies, which are immobilized on a slide. In the case of in situ measurements, the main concern is to ensure the survival of the antibodies under space radiation. Our recent computing simulation of cosmic ray interactions with the martian environment shows that neutrons are one of the dominant species at soil level. Therefore, we have chosen, in a first approach, to study antibody resistance to neutrons by performing irradiation experiments at the Applications Interdisciplinaires des Faisceaux d'Ions en Région Aquitaine (AIFIRA) platform, a French ion beam facility at the Centre d'Etudes Nucléaires de Bordeaux-Gradignan in Bordeaux. Antibodies and fluorescent dyes, freeze-dried and in buffer solution, were irradiated with 0.6 MeV and 6 MeV neutrons. Sample analyses demonstrated that, in the conditions tested, antibody recognition capability and fluorescence dye intensity are not affected by the neutrons.

Light flashbacks during retinal laser photocoagulation following fluorescein angiography.

BACKGROUND AND OBJECTIVE: Yellow light flashbacks can often be seen in cases of laser photocoagulation performed shortly after fluorescein angiography. To determine whether unblocked secondary emission from retained aqueous and vitreous fluorescein may be reaching the treating ophthalmologist, the absorption characteristics of serial dilutions of sodium fluorescein were studied at wavelengths similar to the output of the krypton laser. MATERIALS AND METHODS: A Unicam sp500 series II visible spectrophotometer (Pye Unicam Ltd., Cambridge, UK) was used to measure fluorescein absorption at serial dilutions ranging from 100,000,000 to 100 ng/mL at 488, 518, 568, and 647 nm. sive dilution of fluorescein concentration and with increased wavelength. Blue (488 nm) and green (518 nm) light absorbed at all dilutions of fluorescein. Yellow light (568 nm) did not show any significant absorption below a concentration of 100,000 ng/mL and red light (647 nm) absorbed little below a concentration of 3,000,000 ng/mL. CONCLUSIONS: Dilutions of fluorescein absorbed all commonly used laser wavelengths. There is concern that fluorescein absorption results in emission of light at 520 nm (range: 450 to 700 nm), which may not be blocked by the filters currently placed in fixed laser delivery systems.

Laser-driven acoustic desorption of organic molecules from back-irradiated solid foils.

Laser-induced acoustic desorption (LIAD) from thin metal foils is a promising technique for gentle and efficient volatilization of intact organic molecules from surfaces of solid substrates. Using the single-photon ionization method combined with time-of-flight mass spectrometry, we have examined the neutral component of the desorbed flux in LIAD and compared it to that from direct laser desorption. These basic studies of LIAD, conducted for molecules of various organic dyes (rhodamine B, fluorescein, anthracene, coumarin, BBQ), have demonstrated detection of intact parent molecules of the analyte even at its surface concentrations corresponding to a submonolayer coating. In some cases (rhodamine B, fluorescein, BBQ), the parent molecular ion peak was accompanied by a few fragmentation peaks of comparable intensity, whereas for others, only peaks corresponding to intact parent molecules were detected. At all measured desorbing laser intensities (from 100 to 500 MW/cm2), the total amount of desorbed parent molecules depended exponentially on the laser intensity. Translational velocities of the desorbed intact molecules, determined for the first time in this work, were of the order of hundreds of meters per second, less than what has been observed in our experiments for direct laser desorption, but substantially greater than the possible perpendicular velocity of the substrate foil surface due to laser-generated acoustic waves. Moreover, these velocities did not depend on the desorbing laser intensity, which implies the presence of a more sophisticated mechanism of energy transfer than direct mechanical or thermal coupling between the laser pulse and the adsorbed molecules. Also, the total flux of desorbed intact molecules as a function of the total number of desorbing laser pulses, striking the same point on the target, decayed following a power law rather than an exponential function, as would have been predicted by the shake-off model. To summarize, the results of our experiments indicate that the LIAD phenomenon cannot be described in terms of simple mechanical shake-off or direct laser desorption. Rather, they suggest that multistep energy-transfer processes are involved. Two possible (and not mutually exclusive) qualitative mechanisms of LIAD that are based on formation of nonequilibrium energy states in the adsorbate-substrate system are proposed and discussed.

Photolysis of caged phosphatidic acid induces flagellar excision in Chlamydomonas.

Phosphatidic (PtdOH) acid formation is recognized as an important step in numerous signaling pathways in both plants and mammals. To study the role of this lipid in signaling pathways, it is of major interest to be able to increase the amount of this lipid directly. Therefore, "caged" PtdOH was synthesized, which releases the biologically active PtdOH upon exposure to UV. Analysis of the product revealed that two 2-nitrophenylethyl (NPE) caging groups were coupled to the phosphate headgroup of PtdOH. To measure the quantum efficiency of uncaging, a fluorimetric assay, based on the notion that the NPE cage is an efficient quencher of pyrene fluorescence, was developed. Consequently, after NPE-caged PtdOH and (N-pyrene)-PtdEtn had been mixed in DOPC vesicles, the extent of photolysis of caged PtdOH can be quantified by monitoring the increase in pyrene fluorescence. Using this assay, a quantum yield of 9.6% was determined for the uncaging reaction. The swimming green alga Chlamydomonas moewusii deflagellates upon addition of PtdOH. This response was used to study the release of PtdOH in vivo. Algae incubated with caged PtdOH only arrested swimming after exposure to UV, indicative of PtdOH release. This effect was not observed in the absence of the caged compound or when a control caged compound (caged acetic acid) was added. Fluorescein diacetate staining was used to show that the cells remained viable after UV exposure. The anticipated effect of PtdOH release is confirmed by phase contrast images of UV-exposed algae showing excision of flagella. Together, these results show that caged PtdOH can be used to efficiently increase PtdOH levels, demonstrating that it is a promising precursor for studying PtdOH-dependent signaling.

Two-photon interactions at single fluorescent molecule level.

Single-molecule spectroscopy and single-molecule detection are emerging areas that have many applications when combined with scanning, imaging, and spectroscopy techniques. We have combined a commercial confocal scanning head, to a Ti:sapphire laser and to an inverted microscope, for the detection of single molecule fluorescence of varies dyes by two-photon excitation. We collected spot images of fluorescent molecules that have been deposited on a substrate considering both blinking and photobleaching behavior of fluorescent spots. Here, we report data related to two-photon interactions that occur with the following fluorescent molecules: Indo-1, Rhodamine 6G, Fluorescein, and Pyrene. The choice of these specific dyes is based upon their wide use in biological and medical applications together with the varying complexity of their chemical structure that increases from Pyrene to Indo-1. Moreover, we report some data about single molecule studies related to denaturation of an enhanced green fluorescent protein, GFPmut2, under one photon excitation regime, that show a very similar trend to that observed for the already mentioned fluorescent molecules.

Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements.

Two-photon excitation makes it possible to excite molecules in volumes of much less than 1 fl. In two-photon flash photolysis (TPFP) this property is used to release effector molecules from caged precursors with high three-dimensional resolution. We describe and examine the benefits of using TPFP in model solutions and in a number of cell systems to study their spatial and temporal properties. Using TPFP of caged fluorescein, we determined the free diffusion coefficient of fluorescein (D=4 x 0(-6) cm(2)/s at 20 degrees C, which is in close agreement with published values). TPFP of caged fluorescein in lens tissue in situ revealed spatial nonuniformities in intercellular fiber cell coupling by gap junctions. At the lens periphery, intercellular transport was predominantly directed along rows of cells, but was nearly isotropic further from the periphery. To test an algorithm aiming to reconstruct the Ca(2+) release flux underlying physiological Ca(2+) signals in heart muscle cells, TPFP of DM-Nitrophen was utilized to generate artificial microscopic Ca(2+) signals with known underlying Ca(2+) release flux. In an experiment with mouse oocytes, the recently developed Ca(2+) cage dimethoxynitrophenyl-ethyleneglycol-bis-(beta-aminoethylether)-N,N,N('),N(') tetraacetic acid-4 (DMNPE-4) was released in the oocyte cytosol and inside a nucleolus. Analysis of the resulting fluorescence changes suggested that the effective diffusion coefficient within the nucleolus was half of that in the cytosol. These experiments demonstrate the utility of TPFP as a novel tool for the optical study of biomedical systems.