Nonlinear imaging microscopy for assessing structural and photochemical modifications upon laser removal of dammar varnish on photosensitive substrates.

The nonlinear optical microscopy (NLM) modalities of Multi-Photon Excited Fluorescence (MPEF) and Third Harmonic Generation (THG) have been combined in this work to characterize as a function of depth with micrometric resolution the type and extent of morphological and photochemical modifications that take place upon ultraviolet (UV) pulsed laser removal of a dammar varnish layer applied on a photosensitive substrate. The latter consists on a layer of the synthetic polymer poly-methyl methacrylate doped with a photosensitizer, the aromatic compound 1,4-di[2-(5-phenyloxazolyl)] benzene, that strongly fluoresces upon UV light illumination. A number of laser conditions for partial or total elimination of the varnish coating were explored, namely different wavelengths (266, 248 and 213 nm) and pulse durations, in the nanosecond, picosecond and femtosecond ranges. Changes in the MPEF signals upon laser ablation of the outermost varnish layer successfully signpost photochemical modifications of the varnish or of the photosensitive under-layer, and their dependence with the laser ablation parameters, i.e., wavelength and pulse duration. In turn, THG signals mark the presence of layer boundaries and the reduction by laser ablation of the thickness of the varnish coating. The obtained MPEF and THG data are complemented by morphological observation by optical microscopy and measurements of laser induced fluorescence and micro-Raman spectra of the samples before and after laser ablation at the selected laser irradiation conditions. The results acquired through these non-destructive NLM imaging techniques serve to understand the phenomena that are induced upon laser ablation and to determine the best operating conditions that ensure controlled removal of the varnish with minimal morphological and chemical modifications to the under-layers. This research is of direct application to the UV pulsed laser cleaning of paintings and demonstrates the potential of NLM as a novel assessment tool for non-destructive, on line monitoring of the laser cleaning process.

Third Harmonic Generation microscopy as a reliable diagnostic tool for evaluating lipid body modification during cell activation: the example of BV-2 microglia cells.

Nonlinear optical processes have found widespread applications in fields ranging from fundamental physics to biomedicine. In this study, we attempted to evaluate cell activation by using the Third Harmonic Generation (THG) imaging microscopy as a new diagnostic tool. The BV-2 microglia cell line with or without activation by lipopolysaccharide was chosen as a representative biological model. The results showed that THG imaging could discriminate between the control versus activated state of BV-2 cells not only as to THG signal intensity but also as to THG signal area, while verifying once more that the majority of the intracellular detected signal corresponds to lipid bodies. Since THG imaging is a real time, non-destructive modality and does not require any prior cell processing and staining, the results presented here provide an important tool for normal versus activated cell discrimination, which could be proved very useful in the study of inflammation.

Imaging Caenorhabditis elegans embryogenesis by third-harmonic generation microscopy.

In this study, third-harmonic generation (THG) imaging measurements were performed to characterize different developmental stages of the nematode Caenorhabditis elegans (C. elegans) embryos. Femtosecond laser pulses (1028 nm) were utilized for excitation. THG image contrast modality proved as a powerful diagnostic tool, providing valuable information and offering new insights into the complex developmental process of C. elegans embryogenesis.

Second and third harmonic generation measurements of glues used for lining textile supports of painted artworks.

In this study, we present the implementation of non-linear spot measurements for obtaining specific and novel information related to various types of natural and synthetic glues used for lining of painted artworks. Third harmonic generation measurements were employed, in transmission mode, for the accurate and non-destructive thickness detection of lining glues. Furthermore, second harmonic generation signals were collected, in reflection mode, providing complementary information for the discrimination between different types of lining glues.

In vivo imaging of cell morphology and cellular processes in Caenorhabditis elegans, using non-linear phenomena.

We present the detailed imaging of structures and processes of the nematode Caenorhabditis elegans (C. elegans) using non-linear microscopy. Complementary information about the anatomy of the nematode was collected by implementing a combination of two photon excitation fluorescence (TPEF), second and third harmonic generation (SHG and THG) image contrast modes on the same microscope. Three-dimensional (3D) reconstructions of TPEF, SHG and THG images were also performed. Moreover, THG imaging technique has been tested as a potential, novel, non-destructive diagnostic tool for monitoring cellular processes in vivo, such as neuronal degeneration.

In vivo imaging of neurodegeneration in Caenorhabditis elegans by third harmonic generation microscopy.

In this study, neurodegeneration phenomena were investigated, by performing third harmonic generation imaging measurements on the nematode Caenorhabditis elegans, in vivo. The in vivo, precise identification of the contour of the degenerating neurons in the posterior part of the nematode and the monitoring, in real time, of the progression of degeneration in the worm, through third harmonic generation imaging measurements, were achieved. Femtosecond laser pulses (1028 nm) were utilized for excitation. Thus, the THG image contrast modality comprises a powerful diagnostic tool, providing valuable information and offering new insights into morphological changes and complex developmental processes in live biological specimens.

In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy.

In this study, we use combined two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG) and third-harmonic generation (THG) measurements to image cellular structures of the nematode Caenorhabditis elegans, in vivo. To our knowledge, this is the first time that a THG modality is employed to image live C. elegans specimens. Femtosecond laser pulses (1028 nm) were utilized for excitation. Detailed and specific structural and anatomical features can be visualized, by recording THG signals. Thus, the combination of three image-contrast modes (TPEF-SHG-THG) in a single instrument has the potential to provide unique and complementary information about the structure and function of tissues and individual cells of live biological specimens.

Photophysical properties of Hypericum perforatum L. extracts--novel photosensitizers for PDT.

We report the preparation of the methanolic extract (ME), and polar methanolic fraction (PMF) from the plant Hypericum perforatum L. The extracts contain various photosensitizing constituents such as naphthodianthrone derivatives (in 1.37% w/w), and chlorophylls (in 0.08% w/w). Upon light emission these constituents can be activated, providing photodynamic properties to the extracts, and making them a potent, new class, natural photosensitizers for use in photodynamic therapy (PDT), and photodynamic diagnosis (PDD). The absorbance spectra of the extracts are similar to the spectrum of hypericin, the main naphthodianthrone identified within, with two major bands at 548 and 590 nm. The fluorescence spectra in ethanol exhibit two main bands around 595 and 640 nm, in accordance with the spectrum of pure hypericin. The fluorescence intensity of PMF at 595 nm is only eight times less than the intensity of pure hypericin at the same wavelength, even though its hypericin concentration is only 0.57% w/w. The dependence of the PMF fluorescence signal on the pH of the medium, alone and in comparison with the signal of hypericin, has been investigated. PMF signal fades steadily, and smoothly both in acidic, and basic environment.

Fabrication of three-dimensional structures by three-photon polymerization.

We report the fabrication of three-dimensional structures of submicrometer resolution by three-photon polymerization. This resolution has been achieved by polymerizing ORMOCER, a UV photocurable organic-inorganic hybrid material, with an ultrafast laser irradiation at 1028 nm. To our knowledge, this is the first demonstration of three-photon polymerization, a process that may allow the fabrication of components of very high resolution.

Does Hypericum perforatum L. extract show any specificity as photosensitizer for HL-60 leukemic cells and cord blood hemopoietic progenitors during photodynamic therapy?

UNLABELLED: Autologous bone marrow transplantation is a therapeutic modality that increases the survival rates for children with malignancies with poor prognosis but relapse rates are high and attributed partially to the existence of residual malignant cells. Photodynamic treatment (PDT) has been developed among purging strategies. We investigated the effect of the methanolic extract (ME) and its polar methanolic fraction (PMF) of Hypericum perforatum L., as a new photosensitizer for the leukemic cell line HL-60 and cord blood (CB) hemopoietic progenitors as well as the subcellular localization of the photosensitizer. METHODS: ME and PMF were prepared after extraction of the dry herb with methanol (ME), followed by liquid-liquid extraction with petroleum ether (PMF). Cells were incubated with the extracts before irradiation with Nd-Yvo Laser. Various concentrations of PMF or ME as well as irradiation doses were tested. Following irradiation, cell viability was determined by trypan blue in continuous liquid cultures for HL-60 cells and in clonogenic assays for CB cells. The subcellular localization of the photosensitizer was determined by confocal microscopy. RESULTS: Laser photoirradiation in the presence of both PMF and ME induces the killing of HL-60 cells. This effect is dose dependent. No CFU-GM and BFU-E growth was observed from CB mononuclear cells under the tested experimental conditions. Confocal microscopy revealed that the extracts localize mainly in the cytoplasm of the cells. CONCLUSIONS: PDT with both PMF and ME induces the killing of HL-60 leukemic cells and the optimal conditions of treatment were determined. This effect of PDT/PMF was also exerted on CB progenitor cells indicative of the non-selective uptake of the photosensitizer by malignant cells. Though this suggests that PDT/PMF cannot be helpful in autologous bone marrow purging, these novel extracts can however be beneficial in the PDT treatment of tumors given their photostability, low toxicity and low cost.

Optical characterization of thin female breast biopsies based on the reduced scattering coefficient.

One of the main goals in optical characterization of biopsies is to discern between tissue types. Usually, the theory used for deriving the optical properties of such highly scattering media is based on the diffusion approximation. However, biopsies are usually small in size compared to the transport mean free path and thus cannot be treated with standard diffusion theory. To account for this, an improved theory was developed, by the authors, that can correctly describe light propagation in small geometries (Garofalakis et al 2004 J. Opt. A: Pure Appl. Opt. 6 725-35). The theory's limit was validated by both Monte Carlo simulations and experiments performed on tissue-like phantoms, and was found to be two transport mean free paths. With the aid of this theory, we have characterized 59 samples of breast tissue including cancerous samples by retrieving their reduced scattering coefficients from time-resolved transmission data. The mean values for the reduced scattering coefficients of the normal and the tumour tissue were measured to be 9.7 +/- 2.2 cm(-1) and 10.8 +/- 1.8 cm(-1), respectively. The correlation with age was also investigated.

In vitro optical characterization and discrimination of female breast tissue during near infrared femtosecond laser pulses propagation.

Ultrashort infrared laser pulses were transmitted through excised female breast tissue. The resulted signal was recorded by a streak camera with a time resolution of the order of a few ps. Experimental data of the temporal spread of the ultrashort pulse during the transmission through the tissue have been analyzed using the Patterson analytical expression derived from the diffusion theory. This resulted in the calculation of the absorption and reduced scattering coefficients, which are related to the optical characteristics of each type of tissue. The goal of the study was to use the theoretical values of the coefficients to discriminate different kinds of tissue.

Artificial neural networks for discriminating pathologic from normal peripheral vascular tissue.

The identification of the state of human peripheral vascular tissue by using artificial neural networks is discussed in this paper. Two different laser emission lines (He-Cd, Ar+) are used to excite the chromophores of tissue samples. The fluorescence spectrum obtained, is passed through a nonlinear filter based on a high-order (HO) neural network neural network (NN) [HONN] whose weights are updated by stable learning laws, to perform feature extraction. The values of the feature vector reveal information regarding the tissue state. Then a classical multilayer perceptron is employed to serve as a classifier of the feature vector, giving 100% successful results for the specific data set considered. Our method achieves not only the discrimination between normal and pathologic human tissue, but also the successful discrimination between the different types of pathologic tissue (fibrous, calcified). Furthermore, the small time needed to acquire and analyze the fluorescence spectra together with the high rates of success, proves our method very attractive for real-time applications.

The role of laser-induced fluorescence in myocardial tissue characterization: an experimental in vitro study.

OBJECTIVE: The fluorescence of tissue when stimulated by a laser beam is a well-known phenomenon. The resulting emission spectra depend on the biochemical and structural composition of the tissue. In this study, we examined the spectra of laser-induced fluorescence emitted by myocardial tissue. METHODS: We used an argon-ion laser to stimulate the myocardium of 20 intact sheep hearts. For each spectral emission, we calculated the intensity in specific regions in order to characterize the spectra and to reveal intercavitary and intracavitary morphologic differences. RESULTS: The statistical analysis showed significant differences in the emission spectra intensity between atria and ventricles. The intensity was higher in the atria than in the ventricles (p < 0.001). The atrial emission spectra were morphologically different from those of the ventricles. There was no difference in the intensity or morphology of emission spectra within each chamber. All measurements showed good reproducibility after a short period of time. CONCLUSIONS: Laser-induced fluorescence of myocardial tissue seems to have the characteristics necessary for tissue recognition. This might prove useful in identifying cardiomyopathies and transplant rejection, as well as for myocardial mapping, assisting electrophysiologists in discovering fibrotic arrhythmogenic foci.

Single and double wavelength excitation of laser-induced fluorescence of normal and atherosclerotic peripheral vascular tissue.

Laser-induced fluorescence spectra were recorded from the exposure of peripheral vascular tissue to both helium-cadmium and argon-ion laser radiation. Spectral analysis was based on simple algebraic expressions constructed using the intensity difference of the various spectral regions. The above methods were developed in order to determine the degree of atherosclerosis according to the laser-induced fluorescence signal. Similar results with single wavelength excitation were observed during in vivo irradiation of peripheral vessels.

Photon statistics of laserlike emission from polymeric scattering gain media.

The coherent properties of the temporally and spectrally narrowed emission of laser-induced fluorescence of organic dyes hosted inside artificial scattering matrices (random lasers) were investigated. The excitation source was a frequency-doubled 200-fs pulsed laser emitting at 400 nm. Spectral and temporal features were simultaneously recorded with a spectrograph and a streak camera operating in photon-counting mode. Photon-number distributions were thus created. The temporal coherence of the laserlike emission above and below the excitation energy threshold was investigated from the photon-number distribution that was obtained.

Investigation of the laserlike behavior of polymeric scattering gain media under subpicosecond laser excitation.

The narrowing effects of scatterers on the lifetime and the spectral width of the laser-induced fluorescence of organic dyes hosted in poly(methyl methacrylate) polymer sheets were studied. The excitation source was a distributed-feedback dye laser emitting 0.5-ps pulses at 496 nm. Spectral and temporal features were recorded simultaneously on a spectrograph-streak-camera detection system. The results were then compared with those obtained from dye solutions in methanol that were recorded in previous studies. The effects of the different host environments on the fluorescence characteristics of the dye were thus investigated. These effects are currently studied when the dye is inserted into human tissue in an attempt to boost tumor detection and photodynamic-therapy efficiency. Some initial results are presented.

Effect of liquid-nitrogen and formalin-based conservation in the in vitro measurement of laser-induced fluorescence from peripheral vascular tissue.

In order to investigate the effects of conservation in liquid nitrogen and formalin on peripheral vascular tissue (abdominal aortic, femoral, flank, ham, fibular and tibial artery tissue), laser-induced fluorescence spectra have been recorded during the exposure of these tissues to helium-cadmium and argon ion radiation. The spectral distribution of tissue fluorescence allows the development of simple algorithms based on the intensity difference in order to discriminate the tissue samples when they are fresh and after they have been stored for 24 and 28 h in liquid nitrogen or formalin.

A holographic study of variations in bone deformations resulting from different headgear forces in a macerated human skull.

The effects of headgear on maxillary displacement and the resulting growth modifications are not completely understood, especially regarding the complex relationships between initial and secondary skeletal reactions on one hand and the influence of the direction and magnitude of the applied force on the other. The aim of the present investigation was to study, by means of holographic interferometry, the initial bone displacement occurring in response to headgear traction applied at different force magnitudes and in different directions. Orthopedic forces of 560 grams and orthodontic forces of 354 grams were stimulated on a macerated human skull. The forces came from from high-, straight-, and low-pull headgear traction directed above, through, and below the center of resistance of the maxillary first permanent molars. Immediate skeletal changes were recorded by laser holography. Initial displacements of the maxilla and zygomatic arch in both horizontal and vertical planes were evaluated on frontal and lateral holograms. In most cases, both force magnitudes caused substantial displacements in both planes, albeit to different extents. Complex bending, and rotational, translational, and relative displacements were observed. The direction of displacement did not strictly coincide with that of the applied force. The results of this study indicate that both orthodontic and orthopedic headgear traction may lead to complex initial three-dimensional skeletal displacement in directions not always corresponding with the direction of the applied force.

[A holographic study to demonstrate the initial displacements of a macerated human skull under the influence of the orthodontic force from headgear with traction in different directions]. / Holographische Untersuchung zur Darstellung initialer Verformungen eines mazerierten menschlichen Schädels unter der Einwirkung einer orthopädischen Kraft durch einen Headgear mit verschiedenen Zugrichtungen.

The phenomena of maxillary displacement and the subsequent growth modifications which result from the application of orthopedic headgear traction are very complex and not yet completely understood. The aim of this study was to investigate with the help of holographic interferometry the influence of the headgear's differing traction directions on the initial bone displacement in the maxillary complex. 560 g high, straight, and low pull headgear traction exerted above, through, and below the center of resistance of the upper first permanent molars was simulated on a macerated human skull and skeletal changes were recorded by laser holography as they occurred. On the basis of the frontal and lateral holograms thus obtained, the initial displacement of the maxilla, zygoma, and zygomatic arch in both horizontal and vertical planes was evaluated. Substantial displacements in various parts of the studied structures, displacements such as bending, relative deformation, rotation, and translation, were detected, and, further, the direction of this complex displacement often deviated from the direction of the influencing force. The results obtained by this study indicate that the initial 3-dimensional skeletal displacements brought about by the headgear are very complex. The clinical implication is that they do not always correspond with the direction intended by the force applied.