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Time-resolved fluorescence spectroscopy and imaging was performed on fullerol incorporated in human lens and RPE cells after incubation at doses in the range 1-500 µM to investigate correlation with intracellular distribution and toxicity.

Fast 3-D reconstruction method, based on structured light, has been demonstrated and experimentally validated. Spatial information, resolution and selection of the optimal spatial frequencies are discussed.

Diffuse spectroscopy of turbid media has assumed a crucial role in the characterization of biological tissues. In particular, broadband time-resolved optical spectroscopy allows the direct determination in a single measurement of both the optical parameters of the tissue and the concentration of its main constituents. Moreover, the possibility of performing parallel wavelength measurements allows the recording of data in real time, providing a system that is able to perform dynamic measurements. We used a white-light time-resolved spectroscopy system to monitor absorption changes in a layered diffusive medium. Measurements were performed in reflectance geometry, with a 2-cm sourcedetector distance, on a two-layer liquid phantom with optical properties similar to those of human tissues. By varying the concentrations of three inks with different spectral features, we changed the absorption coefficient of the layers to mimic functional brain activation and the systemic response in the scalp. Data were analyzed by a time-resolved spectrally constrained fitting method based on a homogeneous model of photon diffusion. Although this approach is based on a homogeneous model and employs a single sourcedetector distance, the technique is able to monitor changes in the lower layer, while it is scarcely affected by variations in the upper layer. These results were confirmed by numerical simulations based on a perturbation approach to diffusion theory. Preliminary in vivo measurements have been performed on healthy volunteers to monitor oxy- and deoxy-hemoglobin changes in the brain during a motor task. Although the overall sensitivity of the technique is reduced, in vivo results are in general agreement with the findings of the dedicated system for functional brain activity. © 2006 IEEE.

An imaging technique that combines Optical Projection Tomography with ultrafast time gating is presented. The method provides high resolution reconstruction of scattering samples, which is suitable for 3-D imaging of biological models.

The use of structured light for object localization in diffusive media is discussed. The improvement of spatial resolution is shown. Phase detection of the spatially modulated light is proposed as a method for the localization of inclusions in the medium. Fast three-dimensional localization of an absorbing object based on finite-element analysis reconstruction is demonstrated with experimental data.

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.

We have devised and experimentally validated, on tissue-simulating phantoms and in vivo, a time-resolved spectral fitting analysis for direct assessment of chromophore concentrations and scattering parameters. Experimental data have been acquired with a time-resolved broadband system based on supercontinuum light generated in a photonic crystal fiber and a 32 channel Time Correlated Single Photon Counting system. The novel method is more robust than conventional techniques, especially at low signal-to-noise ratio. © 2006 Optical Society of America.

The use of spatially modulated light is finding application in biomedical optics having potential use in imaging and tomography of tissues and small animals. We describe the time-resolved propagation of spatial frequencies in turbid media. We present a set-up based on a ps laser source, spatially modulated by a micro-mirror device and a time-gated intensifier. We discuss the relevant information content that can be useful for imaging of tissues, in terms of the spatial Fourier components of the propagating pulse. We demonstrate that high spatial frequencies appear in the early time-gated signal whereas low frequencies persist for longer times and that the combined use of high spatial frequencies and early time gates can be used to improve the resolution in imaging.

We present an algorithm for simultaneous reconstruction of optical parameters, quantum yield, and life-time in turbid media with embedded fluorescent inclusions. This algorithm is designed in the Fourier domain as an iterative solution of a system of differential equations of the Helmholtz type and does not involve full ill-conditioned matrix computations. The approach is based on allowing the unknown optical parameters, quantum yield, and lifetime to depend on the Fourier spectral parameter. The algorithm was applied to a time-gated experimental data set acquired by imaging a highly scattering cylindrical phantom concealing small fluorescent tubes. Relatively accurate reconstruction demonstrates the potential of the method.

The time-resolved propagation of spatial frequencies in turbid media is investigated. We discuss the relevant information content in terms of the spatial Fourier component of the propagating pulse. We present a method to record and analyze the time-gated spatial frequency response of highly scattering media. We demonstrate that high spatial frequency information appears in the early time-gated signal, whereas low frequencies persist for longer times.

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.

We describe the development of a compact time-resolved system for the measurement of the optical properties of highly scattering media over a bandwidth of 600-1100 nm. The instrument is based on a fiber laser generating supercontinuum radiation, that is spectrally dispersed and used to sequentially illuminate the sample. A single photon avalanche photo-diode in combination with time correlated single-photon counting is used to recover the time-dispersion curve at each wavelength. The calibration of the system and in-vivo applications are shown.

We demonstrate the feasibility of fluorescence imaging experimentally on the basis of a time gating technique completely in the time domain by using a small number of time steps.

Light propagation in dry and wet softwood (silver fir) was investigated experimentally and theoretically. The spatially and time resolved reflectance from softwood was measured. Light propagation was modeled with Monte Carlo simulations considering the microstructure of softwood. By comparing the spatially resolved reflectance we found that all characteristics of the experimentally obtained iso-intensity contour lines were recovered by the theory. In addition, the reduced scattering and the absorption coefficients were determined in the time domain by fitting a solution of the diffusion equation to Monte Carlo simulations and to measurements. Good qualitative agreement was obtained between the experimentally and theoretically derived optical properties.

Application of adjoint time domain methods to the inverse problem in 3D fluorescence imaging is a novel approach. We demonstrate the feasibility of this approach experimentally on the basis of a time gating technique completely in the time domain by using a small number of time windows. The evolution of the fluorescence energy density function inside a highly scattering cylinder was reconstructed together with optical paramaters. Reconstructed energy density was used in localizing two fluorescent tubes. Relatively accurate reconstruction demonstrates the effectiveness and the potential of the proposed technique. (C) 2008 Optical Society of America.

Time-resolved optical spectroscopy technique in the visible/NIR region (700-1080 nm) is employed to characterize wood samples previously degraded and subsequently consolidated by isoeugenol treatment. Preliminary measurements of absorption spectrum of lignin and cellulose are shown.

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.

We used two fluorescence techniques based on the Indocyanine Green contrast agent to study the effectiveness of antiangionenic drugs in mice. To this purpose, the volume of the active vasculature in different tumor models implanted in mice was assessed by means of a low noise fluorescence imaging setup and by a photon counting system working in transmittance geometry. Using a first tumor model (carcinoma MDA-MB-435) we observed that mice treated with a Vascular Disrupting Agent (ZD6126) showed a reduction in fluorescence emission of the contrast agent with respect to control mice. This was a clear indication of the vascular shutdown that took place in tumors. The effectiveness of the treatment was also confirmed by histological sections. Then, in a second experiment we considered a second tumor model (carcinoma 1A9-VS1) overexpressing the Vascular Endotelial Growth Factor (VEGF121), which is used by tumor cells to promote angiogenesis. We measured the Indocyanine Green fluorescence in mice treated with an antioangiogenic drug (Avastin™) and in control mice. In tumors of treated mice we observed an ICG emission lower than the one detected in control mice. This demonstrated that VEGF activity was effectively blocked by the treatment with Avastin. In conclusion, ICG fluorescence provides a simple and reliable way to assess the effectiveness of vascular targeting therapies. Measurements of the fluorescence signal can be repeated every 24 hours, thus allowing oncologists to perform longitudinal studies on the same animals.

An experimental technique which allows one to perform pump-probe transient absorption spectroscopy in real-time is an important tool to study irreversible processes. This is particularly interesting in the case of biological samples which easily deteriorate upon exposure to light pulses, with the formation of permanent photoproducts and structural changes. In particular pump-probe spectroscopy can provide fundamental information for the design of optical chromophores. In this work a real-time pump-probe imaging spectroscopy system has been realized and we have explored the possibility to further reduce the number of laser pulses by using a time-gated camera. We believe that the use of a time-gated camera can provide an important step towards the final goal of pump-probe single shot spectroscopy.

In this work we evaluate a new approach where zero dispersion at pump wavelength is achieved in a high-order mode (normal dispersion in fundamental mode) and we carried out a quantitative analysis to evaluate the output power in the spectral region 400-600 nm for biomedical application.

Can time-resolved, high-resolution data as acquired by an intensified gated CCD camera (ICCD) aid in the tomographic reconstruction of fluorescence concentration? Usually it is argued that fluorescence is a linear process and thus does not require non-linear, time-dependent reconstructions algorithms, unless absorption and scattering coefficients need to be determined as well. Furthermore, the acquisition of a number of time frames is usually prohibitive for fluorescence measurements, at least in small animals, due to the increased total measurement time. On the other hand, it is obvious that diffusion is less pronounced in images at early gates, due to selective imaging of photons of lower scatter order. This will be the case also for photons emitted by fluorescent sources. Early-gated imaging might increase the contrast in acquired images and could possibly improve fluorescence localization. Herein, we present early gated fluorescence images obtained from phantoms and compare them to continuously acquired data. Increased contrast between background and signal maximum can be observed in time-gated images as compared to continuous data. To make use of the properties exhibited by early gated frames, it is necessary to use a modified reconstruction algorithm. We propose a variant of the well-known Born approximation to the diffusion equation that allows to take into account single time frames. The system matrix for the time-dependent Born approach is more complex to calculate, however the complexity of the actual inverse problem (and the acquisition times) of single-frame reconstructions remains the same as compared to continuous mode.

We report the development of a compact time-resolved system for the measurement of the optical properties of highly scattering media over a bandwidth of 600-1000 nm. The instrument is based on a fiber laser generating supercontinuum radiation, that is spectrally dispersed and sequentially used to illuminate the sample. A single photon avalanche photo-diode in combination with time correlated single-photon counting is used to recover the time-dispersion curve at each wavelength, both fitted by the diffusion equation. Transmittance measurements performed on calibrated epoxy phantoms and in-vivo on female breast are presented, showing good agreement with previous reports. (C) 2007 Optical Society of America.

We demonstrate the feasibility of white-light time-resolved optical mammography. The instrumentation is based on supercontinuum light generated in photonic crystal fiber and 32-channel parallel time-correlated single-photon-counting detection. Total measurement time is of the order of 10 min for typical clinical applications. Preliminary measurements performed on volunteers show the ability of the system to determine tissue constituent concentrations and structure over the entire breast area. Furthermore, measurements on a tissue-like sample demonstrate detection and characterization of inclusions. © 2006 Society of Photo-Optical Instrumentation Engineers.

Profiles of nanometric aluminum and parylene foils have been characterized by soft x-ray contact imaging using a laser-plasma source and a LiF crystal as detector. Due to the characteristic emission of this source in a 2 pi angle, it was possible to obtain the sample image in a wider field of view with respect to coherent sources. LiF crystal is a cheap and robust imaging detector for soft x-ray radiation, that allows one to get high spatial resolution images of thin films with thickness from hundreds down to a few tens of nanometers. (c) 2006 American Institute of Physics.

We present a comparative experimental phantom study of fiber-based and noncontact fluorescence tomography with respect to quantitation and localization of reconstructed fluorescent inclusions in turbid media such as tissue. Noncontact acquisition is usually considered potentially superior to fiber-based techniques because of the availability of a large number of detector readouts through a CCD. Our results indicate, however, that noncontact acquisition itself might improve the quality of reconstructions significantly, even without increasing the number of detectors and thus keeping the inverse problem moderately complex. (c) 2006 Optical Society of America.

We present a detailed characterization of a system for fast time-resolved spectroscopy of turbid media based on supercontinuum generation in a photonic crystal fiber. The light source provides subpicosecond pulses in the 550–1000-nm spectral range, at 85 MHz, at an average power of up to 50 mW. Wavelengthresolved detection is accomplished by means of a spectrometer coupled to a 16-channel, multianode photomultiplier tube, giving a resolution of 4.5–35 nm channel, depending on the grating. Timedispersion curves are acquired with time-correlated single-photon counting, and absorption and reduced scattering coefficients are determined by fitting the data to the diffusion equation. We characterized the system by measuring the time-resolved diffuse reflectance of epoxy phantoms and by assessing the performance in terms of accuracy, linearity, noise sensitivity, stability, and reproducibility. The results were similar to those from previous systems, whereas the full-spectrum (610 810 nm) acquisition time was as short as 1 s owing to the parallel acquisition. We also present the first in vivo real-time dynamic spectral measurements showing tissue oxygenation changes in the arm of a human subject. OCIS codes: 120.6200, 170.6510, 170.7050, 300.6500.

We report on an instrument for time-resolved spectroscopy (TRS) based on white-light generation in a highly non-linear crystal fiber. TRS in the visible and near-infrared region at picosecond-to-nanosecond time scales has attracted increased interest in recent years owing to the possibility of spectroscopic analysis of turbid media, such as biological tissues. A self-mode-locked Ti:Sapphire oscillator pumped by an Ar:ion laser provides pulses 50 - 100 fs long, at 85 MHz repetition rate. The light is focused into a crystal fiber, which consists of a core surrounded by a mesh of air-filled holes. White light is generated by a combination of several non-linear effects in the fiber. We optimize the spectrum for measurements in the region 600 - 1000 nm. For detection, we use an imaging spectrometer coupled to a 16-channel photomultiplier tube, enabling simultaneous detection in 16 wavelength bands. We use time-correlated single-photon counting to record the signal, with a temporal resolution of ~160 ps. To demonstrate the system, we have performed measurements of the diffuse time-resolved reflectance of tissue phantoms made of epoxy resin with added scattering and absorbing materials. The data was evaluated using a light propagation model based on diffusion theory, to extract the scattering and absorption coefficients of the medium. The results corresponded very well with previous measurements on the phantoms performed using other TRS instruments.

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 and0.8 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.

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. © 2005 IOP Publishing Ltd.

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.

Even though absolutely noninvasive techniques are ideally required for the analysis of valuable works of art, in some cases, the historical study or the restoration would benefit from deep investigations that require some kind of alterations on the sample. This is the case when a stratigraphic analysis of a paint has to be carried out to determine pigments and ligants used by the painter. To perform this task, a fully automated device working in situ without physical contact with the sample is certainly the best choice in terms of minimal damage, ease of operation, and quickness of analysis. Such a device could result from the combination of the state of the art portable x-ray fluorescence (XRF) analyzer with a digging tool capable of micrometric control of positioning and depth of operation. In this article, we propose a new method for the determination of chemical element distribution in multilayer samples, based on the use of an alternate sequence of laser photoablations and XRF analysis. The laser sou...

Optical imaging through turbid media has gathered a great interest as a non-invasive method to localize tumors inside the body. A step forward in this direction could be provided by a parallel detector that allows one to acquire a huge amount of data in a short time. This could result in a more stable reconstruction of the optical properties of the sample and in a more reliable diagnosis. We used a system based on a fast gated intensified CCD camera capable of a gate width

Fluorescence lifetime imaging is a rather new and effective tool that can be used to study complex biological samples, either at microscopic or macroscopic levels. The map of the fluorescence lifetime allows one to discriminate amongst different fluorophores and to achieve valuable insights into the behaviour of emitting molecules, leading to information like local pH, oxygen concentration in cells, etc. Moreover, the distribution in space of any fluorescent marker achievable with this technique can be exploited for diagnostic purposes in medicine. After a brief introduction on the motivations for applying fluorescence lifetime imaging in biology and medicine, the basic principles of this technique will be addressed. Then, the two possible implementations of fluorescence lifetime imaging (i.e. the frequency domain and the time domain methods) will be presented. For this purpose, special attention will be devoted to practical aspects of image acquisition and processing, especially for what concerns the time domain method. Then, the analysis of the state-of-the-art systems will include a brief discussion on new concepts that have recently been introduced in this research field. Finally, two interesting applications of fluorescence lifetime imaging will be presented. The former refers to skin tumour detection and has been successfully applied in a preliminary clinical trial, the latter regards DNA chips reading and has been tested only at laboratory level, yet it has produced promising results for its future implementation in commercial systems.

A noninvasive method to assess the local monomer concentration within a wooden matrix, post monomer impregnation, by time-resolved diffuse optical spectroscopy is demonstrated. A data analysis technique for improving accuracy, which takes account of changes in the refractive index during the monomer uptake, has been employed. This technique can be potentially applied in the wood industry for the study of polymer composites as well as in cultural heritage science for noninvasively monitoring the penetration of chemical compounds used for consolidation or conservation purposes.

A new instrumentation based on an intensified CCD camera with picosecond time resolution has been developed for optical imaging in turbid media. Scattering inclusions in a homogeneous sample have been detected by fitting the experimental data with a theoretical expression derived from the random walk theory, while absorption inclusions have been localized using time-gated images taken at suitable delays with respect to the expected arrival time of non-scattered photons.

Time-resolved reflectance spectroscopy was performed on tumor-bearing mice, administered with disulphonated aluminum phthalocyanine (AlS 2 Pc, 5 mg/kg body weight), before, during and after photodynamic therapy. This allowed us to evaluate the absorption spectrum of AlS 2 Pc in vivo from 610 to 700 nm, and to investigate how the therapeutic irradiation affects it. Two tumor locations (intraderma on the back and intramuscular in the leg), and two uptake times (3 and 12 h) were considered. As already observed previously, the absorption spectrum of AlS 2 Pc in vivo is centered at 680–685 nm. The irradiation causes a blue-shift of the measured line shape, more or less marked depending on the experimental conditions. A reduction in absorption is also often observed upon illumination with therapeutic light doses.

In this work time-resolved reflectance spectroscopy (TRS) measurements were performed in vivo on biological tissues (arm and forehead) over a broad spectral range (610-1000 nm) and at different interfiber distances (2-4 cm), by a fully automated system based on mode-locked, continuously tunable laser sources and on time-correlated single photon counting for detection. Preliminary estimates of average tissue optical properties are obtained by interpreting the TRS curves at each wavelength with a homogeneous model for photon migration. The scattering coefficient decreases progressively upon increasing wavelength, while the absorption line shapes show the typical spectral features of the principal tissue components (hemoglobin, water, and lipids), with different weight depending on the tissue type and structure. The best fit of the absorption spectra with the spectra of the pure constituents and of the reduced scattering spectra to Mie theory provided information on the average percentage composition of the different tissues and on the average equivalent tissue structure, respectively. Moreover, TRS measurements at different interfiber distances allowed us to probe different tissue depths, showing the presence of a inhomogeneous structure with different average optical properties.

In this paper we present a system for optical imaging in turbid media based on an intensified CCD camera. The time-gating capability of the light intensifier has been used to acquire time sliced images of the light transmitted through phantoms made of intralipid and ink solutions. Then, we analyzed the images by fitting the time series with a theoretical expression for photon migration. This allowed us to recover the maps of the absorption ((mu) a) and scattering ((mu) s') coefficients in an extended area of the samples. Using our system a possible heterogeneity in the sample can be easily detected. We characterized the performances of the system following two approaches. First, we evaluated the linearity of the relationship between the recovered absorption coefficient and the ink concentration and between the recovered scattering coefficient and the intralipid content (homogeneous samples). Then, we measured the apparent contrast in the (mu) s' maps resulting from small inclusions with a scattering coefficient higher and lower with respect to a homogeneous background (heterogeneous samples). Results encourage further researches.© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Time-resolved reflectance has been used for the nondestructive measurement of optical properties in apples. The technique is based on the detection of the temporal dispersion of a short laser pulse injected into the probed medium. The time distribution of re-emitted photons interpreted with a solution of the diffusion equation yields the mean values of the absorption and reduced scattering coefficients of the medium. The proposed technique proved useful for the measurement of the absorption and scattering spectra of different varieties of apples, revealing the spectral shape of chlorophyll. No major variations were observed in the experimental data when the fruit was peeled, showing that the optical properties measured were those of the pulp. With this technique the change in chlorophyll absorption during storage and ripening could be followed. Finally, a compact prototype working at few selected wavelengths was designed and constructed, demonstrating potentialities of the technique for industrial applications.

A system for time-resolved reflectance spectroscopy was used to measure the absorption spectrum of the breast at different angular positions. Spectra were taken from 615 up to 1000 nm every 5 nm on 8 different locations of the right breast of 2 healthy volunteers. The absorption spectra remarkably changed as a function of positions in both ladies, with major differences among the 2 subjects. A fit of the spectra with the combination of the absorption spectrum of water, lipid, oxy-, and deoxy-hemoglobin permitted to derive the mean concentrations of these main tissue constituents, and to quantify the relative changes as a function of position.

In the present paper we describe a novel approach for DNA microarray reading. We used an intensified CCD camera with picosecond time resolution either for single shot image acquisition or for time resolved measurements. We characterized the time resolution of the system. Then, we demonstrated that two fluorescent markers used for oligonucleotide labeling can be discriminated in the time domain instead of the spectral one. Finally, we applied our system to read a PNA microarray.

A fully automated system for time-resolved reflectance spectroscopy in the wavelength range 610-1010 nm was effectively used to measure the absorption and transport scattering spectra from a two-layer diffusive medium with differences in the absorbing properties. The measured transport scattering spectra at different inter-fibers distances are not influenced by the distinct absorption of the two layers. The measured absorption spectra show a non linear behavior that is strongly dependent on the inter-fiber distance. An algorithm was developed and preliminary applied to reconstruct the absorption spectra of the two layers by exploiting the information obtained from measurements at different inter-fiber distances.

The optical properties (absorption and transport scattering) of biological tissues (arm, abdomen, and forehead) were determined in vivo from 610 to 1010 nm by a fully automated system for time-resolved reflectance spectroscopy, based on a synchronously pumped dye laser (610-695 nm) and an actively mode-locked Titanium.Sapphire laser (700-1010 nm) as light sources and on time-correlated single photon counting for die detection. The scattering decreases progressively upon increasing wavelength, while the absorption line shapes show the typical spectral features of the principal tissue components (hemoglobin, water, and lipids), with different weight depending on the tissue type. The best fit of the absorption spectra with the spectra of the pure constituents and of the transport scattering spectra to Mie theory provided information on the percentage composition of the different tissues and on the internal tissue, respectively.

Time-resolved reflectance and transmittance spectroscopy was used to measure the absorption and transport scattering spectra of the female breast from 610 to 1010 nm in the various phases of the menstrual cycle. This allowed us to investigate how the cycle affects the percentage of water and lipids, and the total hemoglobin content and blood oxygenation of breast tissues. Moreover, the scattering spectra provided information on the tissue structure (i.e., average size of the scattering centers and their concentration).

An imaging system was used to monitor the fluorescence of disulphonated Aluminum Phthalocyanine during the PDT of tumors to check whether an index for therapy effectiveness could be found out. Two excitation wavelengths were tested to discover a possible change in the absorption spectrum of the sensitizer induced by the treatment. Two opposite phenomena take place in the tumor area: a mild photobleaching in the region directly irradiated and a marked increase in the fluorescence signal at the tumor borders. The different behavior observed with the two excitation wavelengths demonstrates that the drug absorption spectrum shifts towards shorter wavelengths as a result of the irradiation.

The optical properties of the breast of a 35 yr. old volunteer were studied as a function of the menstrual period using a system for time resolved reflectance spectroscopy from 610 to 1000 nm. The availability of a wide spectral range allowed us to derive the mean concentration of the main tissue absorbers (water, lipids, oxy-, and deoxy-hemoglobin), and to infer information on the microscopic structure. Reflectance spectra were richer of blood and lipids, while transmittance spectra showed a more marked water peak. Changes of absorption properties were observed as a function of phases within the menstrual cycle with an increase in blood and lipid absorption in reflectance and of water content in transmittance while approaching menstruation. Scattering spectra experienced a drop in slope across ovulation, suggesting a change in size and structure of scatterers. This spectroscopy technique can be of great help to understand breast physiology from an optical point of view, and to help further improvement of optical mammography.

A system for time resolved reflectance and transmittance spectroscopy was developed, providing absorption and scattering spectra of turbid media over a wide spectral range. Critical aspects and technical solutions related to the feasibility of a spectral measurement are discussed. In particular the benefits of using a reference signal recorded during the measurement for the analysis of the experimental data are presented. At the moment the system is unique in terms of covered spectral range and sensitivity.