Frequency offset Raman spectroscopy (FORS) for depth probing of diffusive media.

We present a new technique, frequency offset Raman spectroscopy (FORS), to probe Raman spectra of diffusive media in depth. The proposed methodology obtains depth sensitivity exploiting changes in optical properties (absorption and scattering) with excitation wavelengths. The approach was demonstrated experimentally on a two-layer tissue phantom and compared with the already consolidated spatially offset Raman spectroscopy (SORS) technique. FORS attains a similar enhancement of signal from deep layers as SORS, namely 2.81 against 2.62, while the combined hybrid FORS-SORS approach leads to a markedly higher 6.0 enhancement. Differences and analogies between FORS and SORS are discussed, suggesting FORS as an additional or complementary approach for probing heterogeneous media such as biological tissues in depth.

Diffuse optics using a dual window fast-gated counter.

In this paper we demonstrate the advantages of a fast-gated counter in achieving high count-rate and reducing costs of timing equipment in a time-resolved diffuse optical spectroscopy setup. We experimentally prove the equivalence between the fast-gated counter we developed and a traditional time-correlated single-photon counting setup in terms of depth sensitivity and signal-to-noise ratio. Additionally, we show the suitability of this device for bilayer analysis and to estimate the absorption coefficient of homogeneous diffusing media. Finally, we present a proof-of-principle arterial occlusion measurement on a healthy volunteer to validate the proposed approach in a real application. Fast-gated counters can dramatically reduce both costs and complexity in time-resolved multichannel systems, while achieving high count-rate, thus offering a great advantage in applications like brain and muscle functional imaging.

Recipes to make organic phantoms for diffusive optical spectroscopy.

Three recipes are presented to make tissue constituent-equivalent phantoms of water and lipids. Different approaches to prepare the emulsion are proposed. Nature phantoms are made using no emulsifying agent, but just a professional disperser; instead Agar and Triton phantoms are made using agar or Triton X-100, respectively, as agents to emulsify water and lipids. Different water-to-lipid ratios ranging from 30% to 70% by mass were tested. A broadband time-resolved diffuse optical spectroscopy system was used to characterize the phantoms in terms of optical properties and composition. For some water/lipid ratios the emulsion fails or the phantom has limited lifetime, but in most cases the recipes provide phantoms with a high degree of homogeneity [coefficient of variation (CV) of 4.6% and 1.5% for the absorption and reduced scattering coefficient, respectively] and good reproducibility (CV of 8.3% and 12.4% for absorption and reduced scattering coefficient, respectively).

Reproducibility of identical solid phantoms.

SIGNIFICANCE: Tissue-like solid phantoms with identical optical properties, known within tolerant uncertainty, are of crucial importance in diffuse optics for instrumentation assessment, interlaboratory comparison studies, industrial standards, and multicentric clinical trials. AIM: The reproducibility in fabrication of homogeneous solid phantoms is focused based on spectra measurements by instrument comparisons grounded on the time-resolved diffuse optics. APPROACH: Epoxy-resin and silicone phantoms are considered as matrices and both employ three different instruments for time-resolved diffuse spectroscopy within the spectral range of 540 to 1100 nm. In particular, we fabricated two batches of five phantoms each in epoxy resin and silicone. Then, we evaluated the intra- and interbatch variability with respect to the instrument precision, by considering the coefficient of variation (CV) of absorption and reduced scattering coefficients. RESULTS: We observed a similar precision for the three instruments, within 2% for repeated measurements on the same phantom. For epoxy-resin phantoms, the intra- and the interbatch variability reached the instrument precision limit, demonstrating a very good phantom reproducibility. For the silicone phantoms, we observed larger values for intra- and interbatch variability. In particular, at worst, for reduced scattering coefficient interbatch CV was about 5%. CONCLUSIONS: Results suggest that the fabrication of solid phantoms, especially considering epoxy-resin matrix, is highly reproducible, even if they come from different batch fabrications and are measured using different instruments.

Fast-gated single-photon counting technique widens dynamic range and speeds up acquisition time in time-resolved measurements.

In many time-domain single-photon measurements, wide dynamic range (more than 5 orders of magnitude) is required in short acquisition time (few seconds). We report on the results of a novel technique based on a time-gated Single-Photon Avalanche Diode (SPAD) able to increase the dynamic range of optical investigations. The optical signal is acquired only in well-defined time intervals. Very fast 200-ps gate-ON transition is used to avoid the undesired strong signal, which can saturate the detector, hide the fainter useful signal and reduce the dynamic range. In experimental measurements, we obtained a dynamic range approaching 8 decades in few minutes of acquisition.

Fullerol in human lens and retinal pigment epithelial cells: time domain fluorescence spectroscopy and imaging.

Fullerol is a fullerene derivative that is extensively hydroxylated [nano-C(60)(OH)(24)] and this makes it water-soluble. These fullerene derivatives have shown promise as drug carriers that bypass ocular barriers but fullerols are also potentially phototoxic to human lens and retinal tissues. Fluorescence imaging is a powerful and non-invasive means of probing nanoparticles in biological systems. However, fullerol nanoparticles have a very low level of fluorescence and have not as yet been imaged in vitro and in vivo. Using specialized measurements including time-correlated single photon counting (TCSPC), fullerol fluorescence was determined in aqueous solutions and detected in both human lens and retinal pigment epithelial cells. Time-resolved fluorescence of fullerol (5-200 µM) was characterized in aqueous environment, where the fluorescence decay is best fitted with three lifetimes (3 ns, 0.7-0.9 ns and 0.2 ns). Time-resolved microspectrofluorimetry and time-gated fluorescence imaging were performed on both human lens and retinal pigment epithelial cells incubated with increasing fullerol doses (5-500 µM and 5-50 µM, respectively). Upon increasing concentration, we observe some shortening of the lifetimes, a reduction in the relative amplitude of the shortest-living component and a corresponding increase in the weight of the intermediate-living species. Time-gated imaging of fullerol fluorescence provided information on its intracellular distribution that correlates with progressive cell damage. Therefore time-gated imaging may potentially be used as a means to investigate fullerol distribution and toxicity in the human lens and retina in vivo.

Fluorescence and Fourier-transform infrared spectroscopy for the analysis of iconic Italian design lamps made of polymeric materials.

The preservation of design object collections requires an understanding of their constituent materials which are often polymeric blends. Challenges associated with aging of complex polymers from objects with an unknown physical history may compromise the interpretation of data from analytical techniques, and therefore complicate the assessment of the condition of polymers in indoor museum environments. This study focuses on the analysis of polymeric materials from three well-known Italian design lamps from the 1960s. To assess the degree of chemical modifications in the polymers, non-destructive molecular spectroscopic techniques, Fourier-transform infrared (FTIR) and fluorescence spectroscopy, have been applied directly on the object surfaces using an optical fiber probe and through examination of micro samples. FTIR spectra of the different polymers, polyvinylacetate (PVAc) for the lamps Taraxacum and Fantasma, and both acrylonitrile-butadiene-styrene polymer (ABS) and cellulose acetate (CA) for the lamp Nesso, allowed the detection of ongoing deterioration processes. Fluorescence spectroscopy proved particularly sensitive for the detection of molecular changes in the polymeric objects, as the spectra obtained from the examined lamps differ significantly from those of the unaged reference materials. Differences in fluorescence spectra are also detected between different points on the same object further indicating the presence of different chemical species on the surfaces. With the aid of complementary data from FTIR spectroscopy, an interpretation of the emission spectra of the studied polymeric objects is here proposed, further suggesting that fluorescence spectroscopy may be useful for following the degradation of historical polymeric objects.

In vivo test-driven upgrade of a time domain multi-wavelength optical mammograph.

A recent upgrade of the time domain multi-wavelength optical mammograph developed by Politecnico di Milano achieved good performance in laboratory tests [Biomed. Opt. Express9, 755 (2018).10.1364/BOE.9.000755]. However, it proved unsatisfactory when in vivo measurements were finally performed. That led to a further upgrade, including the replacement of the time-to-digital converter with a new model, and the related set-up changes. The new instrument version offers improved laboratory performance (as assessed through established protocols: BIP and MEDPHOT) and good in vivo performance (extension of the scanned breast area, repeatability, consistency of estimated tissue composition with physiology). Besides introducing the new set-up and detailing its laboratory and in vivo performance, we highlight the importance of systematic in vivo testing before entering clinical trials.

Optical characterization of 3D printed PLA and ABS filaments for diffuse optics applications.

The interest for Fused Deposition Modelling (FDM) in the field of Diffuse Optics (DO) is rapidly increasing. The most widespread FDM materials are polylactic acid (PLA) and acrylonitrile butadiene styrene (ABS), thanks to their low cost and easiness-to-print. This is why, in this study, 3D printed samples of PLA and ABS materials were optically characterized in the range from the UV up to the IR wavelengths, in order to test their possible employment for probe construction in DO applications. To this purpose, measurements with Near Infrared Spectroscopy and Diffuse Correlation Spectroscopy techniques were considered. The results obtained show how the material employed for probe construction can negatively affect the quality of DO measurements.

Optical signatures of radiofrequency ablation in biological tissues.

Accurate monitoring of treatment is crucial in minimally-invasive radiofrequency ablation in oncology and cardiovascular disease. We investigated alterations in optical properties of ex-vivo bovine tissues of the liver, heart, muscle, and brain, undergoing the treatment. Time-domain diffuse optical spectroscopy was used, which enabled us to disentangle and quantify absorption and reduced scattering spectra. In addition to the well-known global (1) decrease in absorption, and (2) increase in reduced scattering, we uncovered new features based on sensitive detection of spectral changes. These absorption spectrum features are: (3) emergence of a peak around 840 nm, (4) redshift of the 760 nm deoxyhemoglobin peak, and (5) blueshift of the 970 nm water peak. Treatment temperatures above 100 °C led to (6) increased absorption at shorter wavelengths, and (7) further decrease in reduced scattering. This optical behavior provides new insights into tissue response to thermal treatment and sets the stage for optical monitoring of radiofrequency ablation.

Time-gated optical projection tomography.

We present an imaging technique that combines optical projection tomography with ballistic imaging using ultrafast time gating. The method provides high-resolution reconstruction of scattering samples and is suitable for three-dimensional (3D) imaging of biological models.

Non-invasive investigation of adipose tissue by time domain diffuse optical spectroscopy.

The human abdominal region is very heterogeneous and stratified with subcutaneous adipose tissue (SAT) being one of the primary layers. Monitoring this tissue is crucial for diagnostic purposes and to estimate the effects of interventions like caloric restriction or bariatric surgery. However, the layered nature of the abdomen poses a major problem in monitoring the SAT in a non-invasive way by diffuse optics. In this work, we examine the possibility of using multi-distance broadband time domain diffuse optical spectroscopy to assess the human abdomen non-invasively. Broadband absorption and reduced scattering spectra from 600 to 1100 nm were acquired at 1, 2 and 3 cm source-detector distances on ten healthy adult male volunteers, and then analyzed using a homogeneous model as an initial step to understand the origin of the detected signal and how tissue should be modeled to derive quantitative information. The results exhibit a clear influence of the layered nature on the estimated optical properties. Clearly, the underlying muscle makes a relevant contribution in the spectra measured at the largest source-detector distance for thinner subjects related to blood and water absorption. More unexpectedly, also the thin superficial skin layer yields a direct contamination, leading to higher water content and steeper reduced scattering spectra at the shortest distance, as confirmed also by simulations. In conclusion, provided that data analysis properly accounts for the complex tissue structure, diffuse optics may offer great potential for the continuous non-invasive monitoring of abdominal fat.

Total synchronous fluorescence spectroscopy combined with multivariate analysis: method for the classification of selected resins, oils, and protein-based media used in paintings.

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.

Seven-wavelength time-resolved optical mammography extending beyond 1000 nm for breast collagen quantification.

Our multi-wavelength time-resolved optical mammograph was upgraded to improve its overall performances and extend its spectral coverage up to 1060 nm, with the aim of increasing the measurement sensitivity to the content of collagen in breast tissue. Late-gated intensity and reduced scattering images are routinely displayed for diagnostic purposes. Maps of tissue constituents (lipid, water and collagen) and blood parameters (total hemoglobin content and blood oxygenation) are built to highlight spatial changes due to physiological and pathological reasons. The upgraded instrument was tested on tissue phantoms. Then images were collected at 7 wavelengths (635-1060 nm) from 10 healthy volunteers. Average collagen content correlated with breast density whenever x-ray mammograms were available (6 subjects).

Effect of prolonged stimulation on cerebral hemodynamic: a time-resolved fNIRS study.

PURPOSE: Sustained attention is one of the most important cognitive abilities for the management of everyday life, but it is often studied only at the behavioral level, while functional correlates are scarcely investigated. In this article, the authors address the topic of characterizing the dynamics of cerebral metabolism in the prefrontal cortex during a task of prolonged attention. METHOD: By means of multichannel time-resolved functional near-infrared spectroscopy and generalized linear model based data processing, the authors measured the hemodynamic response of the prefrontal cortex from 19 healthy subjects to a shortened version of a sustained attention task (Conners' Continuous Performance Test), lasting for 10 min. RESULTS: The task elicited significant brain activation, which did not remain constant for the entire task, but showed a drop not correlated with performance decay 4 min after the beginning of the task. Furthermore, oxygenated hemoglobin showed an increasing trend also during the first phase of the recovery, just after the end of the task. CONCLUSION: The results indicate a nontrivial dynamics of neural activation, habituation processes, and hemodynamic/metabolic coupling. These results encourage further studies about continuous stimulation of cognitive functions on both healthy and pathological subjects.

Assessment of the ageing of triterpenoid paint varnishes using fluorescence, Raman and FTIR spectroscopy.

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.

Combined reconstruction of fluorescent and optical parameters using time-resolved data.

We present an algorithm for simultaneous reconstruction of optical parameters, quantum yield, and lifetime 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.

Assessment of variations in moisture content of wood using time-resolved diffuse optical spectroscopy.

Time-resolved spectroscopy using a pulsed supercontinuum source was employed for the assessment of moisture content (MC) in dry wood. The MC of wood at different relative humidities between 94% and 12% has been monitored. Following curve fitting, absorption spectra between 700 and 1100 nm of samples suggest that differences in the MC of wood are appreciable at around 970 nm. Further, dynamic changes in MC were simulated following a change in relative humidity. Differences in absorption spectra with changes in MC of around 1% can be detected. Various applications of the technique are suggested.

In vivo measurement of vascular modulation in experimental tumors using a fluorescent contrast agent.

We compared the effectiveness of three optical techniques based on fluorescence imaging and spectroscopy with indocyanine green (ICG) contrast agent to evaluate in vivo the disruption of the active vasculature induced by a vascular targeting agent. The blood perfusion of the MDA-MB-435 tumor model transplanted in nude mice was estimated from the signal of the contrast agent measured immediately after its systemic injection in mice. Optical measurements were performed using a fluorescence imaging setup and a fiber-based time correlated single photon counting (TCSPC) apparatus. This latter apparatus was used to measure the tumor fluorescence in transmittance geometry and the change in the basal optical absorption induced by the contrast agent, thus providing an alternative estimation of the blood content in the tumor. Mice were divided into four groups. Three groups were treated with different doses of the vascular disrupting agent ZD6126, the fourth group (control group) received the drug vehicle only. Optical measurements were carried out 3 h after pharmacologic treatment. After 24 h, mice were killed, tumors were excised and the extent of necrosis was evaluated with standard histologic analysis. On fluorescence imaging ICG emission from tumors of mice treated with ZD6126 significantly was lower compared with the emission from control mice. The histologic sections also showed a significantly higher amount of necrosis in tumors of treated mice. Both these findings, which correlate with each other, indicate an effective vascular shutdown induced by the drug. However, ICG fluorescence measured with the TCSPC apparatus in transmittance geometry and the estimate of the change in optical absorption did not allow a statistically significant differentiation between treated and control groups.

Time-resolved scanning system for double reflectance and transmittance fluorescence imaging of diffusive media.

In this work we present a novel diffuse fluorescence imaging system, based on time-resolved two-wavelength double reflectance and transmittance setup for slab geometry samples. We describe the hardware setup, showing its compactness and versatility and show the results on preliminary measurements on phantoms. We fully assessed the performances and the dynamic ranges of the system. We validated its ability of recovering the optical properties of the bulk medium, for samples with scattering and absorption coefficients similar to those of biological tissues and with thicknesses of about 2 cm. Moreover we assess the linearity of the recorded signals against the fluorophore concentration, when it is homogeneously diffused in the phantom or concentrated inside a sealed inclusion. In both cases we observe again a fairly good linearity, over three orders of magnitude, from 10(-8)M to 10(-5)M. With the fluorescent inclusion we were also able to assess the imaging capabilities of the system, in terms of spatial resolution, which we appraise in about 3 mm, and in terms of imaging sensitivity (the smallest quantity of fluorescent dye distinguishable from the homogeneous background), settled to 200 fmol. Since the recorded data are time resolved, we could also estimate the dye fluorescence lifetime and build early and late time gate images. We finally discuss some of the criticalities of the proposed system and the developments we are currently carrying on in order to adapt it for in vivo measurements.