Abstract: This paper discusses the spectral distortions occurring when time-resolved spectroscopy of diffusive media is performed illuminating with a wide bandpass. It is shown that the spectral region within the bandpass that exhibits the lowest absorption will dominate the resulting time-resolved curve, leading to significant underestimations of absorption as well as distortions in the spectral shape (including shifts in peak positions). Due to the nonlinear behavior of absorption, this effect becomes even more pronounced when including longer and longer photon path lengths. First, a theoretical treatment of the problem is given, and then the distortion is described by time-resolved reflectance simulations and experimental measurements of lipid and water samples. A spectrally constrained data analysis is proposed that takes into account the spectrum of the light injected into the sample, used to overcome the distortion and improve the accuracy of the estimation of chromophore concentrations from absorption spectra. Measurements on a lipid sample show a reduction of the error from 30% to 6%.
Abstract: The feasibility of in vivo measurements in the range of 1000 to 1100 nm and the potential benefits of operation in that wavelength range for diagnostic applications are investigated. To this purpose, an existing system for time-resolved diffuse spectroscopy is modified to enable in vivo studies to be carried out continuously from 600 to 1100 nm. The optical characterization of collagen powder is extended to 1100 nm and an accurate measurement of the absorption properties of lipid is carried out over the entire spectral range. Finally, the first in vivo absorption and scattering spectra of breast tissue are measured from 10 healthy volunteers between 600 and 1100 nm and tissue composition is evaluated in terms of blood parameters and water, lipid, and collagen content using a spectrally constrained global fitting procedure.
Abstract: Recent interest in the fluorescence of binding media and varnishes (proteins, oils, and resins) commonly used in paintings is based on the potential for discriminating these organic materials. A useful way of studying the presence of the broad-band fluorescence emissions found in these complex organic materials is fluorescence excitation emission spectroscopy. However, due to the presence of Raman and Rayleigh scattering which may necessitate correction or preprocessing for statistical analysis and visualization, an alternative approach has been adopted for the analysis of different samples of artist materials based on total synchronous fluorescence spectroscopy. Films of selected drying oils, glue, egg, and casein and the resins mastic, dammar, copal, and shellac were analyzed using total synchronous fluorescence spectroscopy, and an interpretation of the differences between spectra is given. A data reduction method based on the transformation of fluorescence contours extracted from total synchronous fluorescence from Cartesian to polar coordinates is presented and is followed by the comparison of data using multivariate analysis and hierarchical cluster analysis. Results suggest that the new method can be used to classify samples on the basis of their fluorescence spectra, clearly differentiating oils, resins, and protein-based media into groups.
Abstract: The assessment of the influence of natural and artificial ageing on the spectrofluorescence of triterpenoid varnishes dammar and mastic is the focus of this work. Both Fourier transform infrared (FTIR) microscopy using attenuated total reflectance and Raman spectroscopy have been employed for complementary molecular analysis of samples. Synchronous fluorescence spectroscopy, excitation emission spectroscopy, and statistical analysis of data have been used to monitor changes in the optical properties of varnish samples. Assessment of naturally and artificially aged samples using excitation emission spectroscopy suggests that extensive exposure to visible light does not lead to easily appreciable differences in the fluorescence of mastic and dammar; cluster analysis has been used to assess changes, which occur with artificial ageing under visible light, indicating that differences in the fluorescence spectra of aged triterpenoids may be insufficient for their discrimination. The results highlight significant differences between the initial fluorescence of films of dammar and mastic and the fluorescence, which develops with ageing and oxidation, and specific markers, which change with ageing in FTIR and Raman spectra, have been identified.
Abstract: The analysis of various varnishes from different origins, which are commonly found on historical musical instruments was carried out for the first time with both fluorescence excitation emission spectroscopy and laser-induced time-resolved fluorescence spectroscopy. Samples studied include varnishes prepared using shellac, and selected diterpenoid and triterpenoid resins from plants, and mixtures of these materials. Fluorescence excitation emission spectra have been collected from films of naturally aged varnishes. In parallel, time-resolved fluorescence spectroscopy of varnishes provides means for discriminating between short- (less than 2.0 ns) and long-lived (greater than 7.5 ns) fluorescence emissions in each of these complex materials. Results suggest that complementary use of the two non destructive techniques allows a better understanding of the main fluorophores responsible for the emission in shellac, and further provides means for distinguishing the main classes of other varnishes based on differences in fluorescence lifetime behaviour. Spectrofluorimetric data and time resolved spectra presented here may form the basis for the interpretation of results from future in situ fluorescence examination and time resolved fluorescence imaging of varnished musical instruments.
Abstract: We have proposed and experimentally demonstrated that picosecond time-resolved optical spectroscopy in the visible/near-infrared (NIR) region (700-1040 nm) is a useful technique for noninvasive characterization of wood. This technique has been demonstrated on both softwood and hardwood samples treated in different ways simulating the aging process suffered by waterlogged woods. In all the cases, alterations of absorption and scattering spectra were observed, revealing changes of chemical and structural composition.
Abstract: Ultraviolet-induced fluorescence spectroscopy is a commonly used technique for the characterization and identification of painting materials, such as organic binders and colorants. Its interpretation is strictly connected to both the experimental setup and an understanding of the physical and chemical interactions among materials in paint layers, which are commonly composed of a fluorescent organic binder and a pigment. When irradiated with ultraviolet radiation, the light emitted by fluorophores present in the organic binder undergoes several types of interactions, in particular scattering and absorption by neighboring pigmented particles and auto-absorption. As a result of scattering and absorption phenomena, the emission spectrum is deformed according to the physical properties of the surrounding pigmented particles. This can lead to shifts of the emission maxima and/or to the formation of apparent new emission bands. The extent of the modifications to the emission spectra, caused by auto-absorption and selective absorption phenomena, may lead to the erroneous characterization or identification of the fluorescent materials. As a consequence, the interpretation of the emission signal can be greatly compromised. A correction based on the Kubelka-Munk theory is proposed to evaluate the extent of the spectral distortion and is assessed on modern replicas of wall paintings of known composition. Although the model cannot be applied to all cases, qualitative distinctions between real and apparent emissions are achieved.
Abstract: A portable fluorescence multispectral imaging system was developed and has been used for the analysis of artistic surfaces. The imaging apparatus exploits two UV lamps for fluorescence excitation and a liquid crystal tunable filter coupled to a low-noise charge coupled device as the image detector. The main features of the system are critically presented, outlining the assets, drawbacks, and practical considerations of portability. A multivariate statistical treatment of spectral data is further considered. Finally, the in situ analysis with the new apparatus of recently restored Renaissance wall paintings is presented.
Abstract: We present an in vivo broadband spectroscopic characterization of the human forehead. Absorption and scattering properties are measured on five healthy volunteers at five different interfiber distances, using time-resolved diffuse spectroscopy and interpreting data with a model of the diffusion equation for a homogeneous semi-infinite medium. A wavelength-tunable mode-locked laser and time-correlated single-photon counting detection are employed, enabling fully spectroscopic measurements in the range of 700-1000 nm. The results show a large variation in the absorption and scattering properties of the head depending on the subject, whereas intrasubject variations, assessed at different interfiber distances, appear less relevant, particularly for what concerns the absorption coefficient. The high intersubject variability observed indicates that a unique set of optical properties for modeling the human head cannot be used correctly. To better interpret the results of the analysis of in vivo measurements, we performed a set of four-layer model Monte Carlo simulations based on different data sets for the optical properties of the human head, partially derived from the literature. The analysis indicated that, when simulated time-resolved curves are fitted with a homogeneous model for the photon migration, the retrieved absorption and reduced scattering coefficients are much closer to superficial layer values (i.e., scalp and skull) than to deeper layer ones (white and gray matter). In particular, for the shorter interfiber distances, the recovered values can be assumed as a good estimate of the optical properties of the first layer.
Abstract: Time-resolved transmittance spectroscopy was performed in the wavelength range of 610 or 700 to 1050 nm on phantom parallelepipeds and bone tissue cubes of different sizes. The data were best fitted with solutions of the diffusion equation for a laterally infinite slab and for a parallelepiped to investigate how size and optical properties of the samples affect the results obtained with the two models. Monte Carlo simulations were also performed to support and help with the interpretation of the experimental data. The parallelepiped model performs much better than the infinite slab model for the estimate of the reduced scattering coefficient and, even more, the absorption coefficient. It can profitably be used to quantify the optical properties of biological tissue samples and to derive information such as tissue composition, when small volumes are involved.
Abstract: The absorption spectrum of collagen powder is measured between 610 and 1040 nm by time-resolved transmittance spectroscopy. Absorption spectra of breast from healthy volunteers are then interpreted, adding collagen to the other absorbers previously considered (i.e., oxy- and deoxyhemoglobin, water, and lipids). A significant amount of collagen, depending on breast type, is estimated to be present. Adding collagen to the fitting procedure affects remarkably the estimated values of blood content and oxygenation. The quantification of collagen has potential implications for the assessment of breast density and cancer risk.
Abstract: We investigate anisotropic light propagation in biological tissue in steady-state and time domains. Monte Carlo simulations performed for tissue that consists of aligned cylindrical and spherical scatterers show that steady-state and time-resolved reflectance depends strongly on the measurement direction relative to the alignment of the cylinder axis. We examine the determination of optical properties using an isotropic diffusion model and find that in the time domain, in contrast to steady-state spatially resolved reflectance measurements, the obtained absorption coefficient does not depend on the measurement direction and is close to the true value. Contrarily, the derived reduced scattering coefficient depends strongly on the measurement direction in both domains. Measurements of the steady-state and time-resolved reflectance from bovine tendon confirm the theoretical findings.
Abstract: The differentiation of proteins commonly found as binding media in paintings is presented based on spectrally resolved and time-resolved laser-induced fluorescence (LIF) and total emission spectroscopy. Proteins from eggs and animal glue were analysed with pulsed laser excitation at 248 nm (KrF excimer) and 355 nm (third harmonic of Nd:YAG) for spectrally resolved measurements, and at 337 nm (N2) and 405 nm (N2 pumped dye laser) for spectrally resolved lifetime measurements and fluorescence lifetime imaging (FLIM). Total emission spectra of binding media are used for the interpretation of LIF spectra. Time-resolved techniques become decisive with excitation at longer wavelengths as fluorescence lifetime permits the discrimination amongst binding media, despite minimal spectral differences; spectrally resolved measurements of fluorescence lifetime have maximum differences between the binding media examined using excitation at 337 nm, with maximum observed fluorescence at 410 nm. FLIM, which measures the average lifetime of the emissions detected, can also differentiate between media, is non-invasive and is potentially advantageous for the analysis of paintings.
Abstract: We describe a system for absorption and scattering spectroscopy of diffusive media based on time-resolved reflectance and transmittance measurements. The system is operated with mode-locked lasers tunable in the 550-1050 nm spectral range and on a detection chain based on time-correlated single-photon counting. All measurement procedures such as laser tuning and optimization, signal conditioning, data acquisition, and analysis are completely automated, permitting spectral measurements over the whole range in a few minutes. The criticalities of the system are discussed together with the strategies to compensate them. The Medphot protocol devised for the characterization of photon migration instruments was applied to assess the system performances in terms of accuracy, linearity, noise, stability, and reproducibility. Finally, an example of application of the instrument to the spectroscopy of powders is presented.
Abstract: A set-up, based on a CCD camera, to localize fluorescent inclusions in diffusing media was developed. This set-up allows one to acquire a huge dataset along two axes. This aspect is fundamental to performing a tomographic reconstruction in order to quantify the fluorescence amplitude in each voxel of the sample. Firstly, a simple analytical approach to recover the position of a single inclusion, embedded in a turbid medium, was developed. Then, we implemented a reconstruction algorithm to recover the position of one and two inclusions and to estimate their relative concentrations. Finally, we studied the dependence of reconstructed data on the number of injection points of excitation light and the number of detection points of fluorescence emission.
Abstract: We developed a combined procedure for the analysis of works of art based on a portable system for fluorescence imaging integrated with analytical measurements on microsamples. The method allows us to localize and identify organic and inorganic compounds present on the surface of artworks. The fluorescence apparatus measures the temporal and spectral features of the fluorescence emission, excited by ultraviolet (UV) laser pulses. The kinetic of the emission is studied through a fluorescence lifetime imaging system, while an optical multichannel analyzer measures the fluorescence spectra of selected points. The chemical characterization of the compounds present on the artistic surfaces is then performed by means of analytical measurements on microsamples collected with the assistance of the fluorescence maps. The previous concepts have been successfully applied to study the contaminants on the surface of Michelangelo's David. The fluorescence analysis combined with Fourier transform infrared (FT-IR) measurements revealed the presence of beeswax, which permeates most of the statue surface, and calcium oxalate deposits mainly arranged in vertical patterns and related to rain washing.
Abstract: A system based on a picosecond time-gated image intensifier is proposed for non-contact testing of CMOS circuits. The apparatus allows one to record the temporal evolution of the luminescence emitted during transistor switching as a function of the position inside the chip. The system is characterized by an intrinsic parallelism in the spatial dimensions. This feature is noticeable for studying wide sections of complex circuits, like microprocessors and random access memories, where multiple electrical events occur simultaneously. Experiments on a CMOS inverter chain and on a static memory have been carried out, in order to demonstrate the applicability of a picosecond time-gated imager to circuit analysis.
Abstract: Hypericin is the active ingredient of the off-the-shelf antidepressant St. John's Wort. It is an effective phototoxic agent and its systemic administration at therapeutic doses could induce particular damage in the eye due to continuous light exposure. Hypercin is strongly fluorescent and its fluorescence properties can be monitored to investigate noninvasively its localization and interactions. To this aim, time-resolved microspectrofluorimetry and fluorescence lifetime imaging were used to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by retinal pigment epithelium (RPE) cells treated with Hyp at concentrations in the micromolar range (0.5-10 microM). In the presence of hypericin, the emission peaks at 600-605 nm and the fluorescence decay is best fitted with three lifetimes (5.5-7 ns, 1.9-2.5 ns and <0.8 ns). Spectral and temporal differences were observed between high (> or =5 microM) and low hypericin concentrations. In particular, upon increasing concentration, the emission spectrum of the slow component broadens and its lifetime shortens. The latter change is observed also when high concentrations are reached locally, due to more efficient localization within the cell.
Abstract: A system for advanced fluorescence investigation of works of art has been assembled and integrated in a characterization procedure that allows one to localize and identify organic compounds that are present in artworks. At the beginning of the investigation, fluorescence lifetime imaging and spectroscopy address a selective microsampling of the artwork. Then analytical measurements of microsamples identify the chemical composition of the materials under investigation. Finally, on the basis of fluorescence lifetime and amplitude maps, analytical data are extended to the whole artwork. In such a way, information on the spatial distribution of organic materials can be inferred. These concepts have been successfully applied in an extensive campaign for analysis of Renaissance fresco paintings in Castiglione Olona, Italy. Residue of various types of glue and stucco left from a restoration carried out in the early 1970s was localized and classified. Insight into the technique used by the painter to make gilded reliefs was also obtained.
Abstract: Different approaches for absorption and scattering spectroscopy of living tissues are discussed. In particular, a unique system for time-resolved reflectance and transmittance spectroscopy is presented, capable of acquiring in vivo absorption and scattering spectra of diffusive media between 600 and 1000 nm. A review of typical spectra obtained from a variety of tissue structures is shown, including female breast, forearm, abdomen, and forehead. A second-level analysis of the measured spectra permits an estimation of the concentrations of the key tissue absorbers, as well as of the Mie-equivalent scattering radii. Further, absorption and scattering spectra can be used to estimate the penetration depth of light in tissues as a function of wavelength, which is a crucial parameter in view of the possible application of optical in vivo molecular imaging in clinical diagnosis. Finally, an example of the applicability of the methodology to other biological media such as fruits and vegetables is shown.
Abstract: We describe a novel technique for DNA-microarray reading based on time-resolved fluorescence measurements. We used an intensified CCD camera with picosecond resolution to acquire a set of time-delayed fluorescence images from a mutation DNA microarray marked with cyanine 3. We measured the fluorescence lifetimes of the marker and the background separately, and we used this information to calculate the amplitude map of the marker, starting from the time-delayed images. This procedure allowed us to identify hybridized spots that are not visible in fluorescence images acquired with continuous-wave detection.
Abstract: A fluorescence imaging system was used to monitor the emission of disulfonated aluminum phthalocyanine (AlS2Pc) during the photodynamic therapy (PDT) of murine tumors. Cells of the MS-2 fibrosarcoma were injected in mice in two compartments in order to cause the development of tumors in different host tissues. Two drug doses and two uptake times were considered. Moreover, the fluorescence of the AlS2Pc was excited using two wavelengths on the opposite sides of the absorption peak to detect a possible change in the absorption spectrum of the sensitizer induced by the PDT. In the tumors, the treatment induces a variation of the fluorescence intensity: in some mice a mild photobleaching takes place, in others a fluorescence enhancement occurs. Which effect predominates depends on the experimental conditions, even though a large spread of data was found amongst mice of the same group. In all mice, independently of the drug dose, uptake time or tumor compartment, a marked increase in the fluorescence signal takes place at the borders of the irradiated area. To quantify this effect we evaluated the ratio between the fluorescence intensities in the peritumoral area and in the tumor itself. This ratio increases monotonically during the PDT, showing a different behavior with the two excitation wavelengths. This indicates that the AlS2Pc absorption spectrum shifts toward shorter wavelengths as a result of the irradiation.
