Optical signatures of thermal damage on ex-vivo brain, lung and heart tissues using time-domain diffuse optical spectroscopy.

Thermal therapies treat tumors by means of heat, greatly reducing pain, post-operation complications, and cost as compared to traditional methods. Yet, effective tools to avoid under- or over-treatment are mostly needed, to guide surgeons in laparoscopic interventions. In this work, we investigated the temperature-dependent optical signatures of ex-vivo calf brain, lung, and heart tissues based on the reduced scattering and absorption coefficients in the near-infrared spectral range (657 to 1107 nm). These spectra were measured by time domain diffuse optics, applying a step-like spatially homogeneous thermal treatment at 43 °C, 60 °C, and 80 °C. We found three main increases in scattering spectra, possibly due to the denaturation of collagen, myosin, and the proteins' secondary structure. After 75 °C, we found the rise of two new peaks at 770 and 830 nm in the absorption spectra due to the formation of a new chromophore, possibly related to hemoglobin or myoglobin. This research marks a significant step forward in controlling thermal therapies with diffuse optical techniques by identifying several key markers of thermal damage. This could enhance the ability to monitor and adjust treatment in real-time, promising improved outcomes in tumor therapy.

Initial non-invasive in vivo sensing of the lung using time domain diffuse optics.

The in vivo diagnosis and monitoring of pulmonary disorders (caused for example by emphysema, Covid-19, immature lung tissue in infants) could be effectively supported by the non-invasive sensing of the lung through light. With this purpose, we investigated the feasibility of probing the lung by means of time-resolved diffuse optics, leveraging the increased depth (a few centimeters) attained by photons collected after prolonged propagation time (a few nanoseconds). We present an initial study that includes measurements performed on 5 healthy volunteers during a breathing protocol, using a time-resolved broadband diffuse optical spectroscopy system. Those measurements were carried out across the spectral range of 600-1100 nm at a source-detector distance of 3 cm, and at 820 nm over a longer distance (7-9 cm). The preliminary analysis of the in vivo data with a simplified homogeneous model revealed a maximum probing depth of 2.6-3.9 cm, suitable for reaching the lung. Furthermore, we observed variations in signal associated with respiration, particularly evident at long photon propagation times. However, challenges stemming from both intra- and inter-subject variability, along with inconsistencies potentially arising from conflicting scattering and absorption effects on the collected signal, hindered a clear interpretation. Aspects that require further investigation for a more comprehensive understanding are outlined.

Statistics of maximum photon penetration depth in a two-layer diffusive medium.

We present numerical results for the probability density function f(z) and for the mean value of photon maximum penetration depth zmax> in a two-layer diffusive medium. Both time domain and continuous wave regime are considered with several combinations of the optical properties (absorption coefficient, reduced scattering coefficient) of the two layers, and with different geometrical configurations (source detector distance, thickness of the upper layer). Practical considerations on the design of time domain and continuous wave systems are derived. The methods and the results are of interest for many research fields such as biomedical optics and advanced microscopy.

Application limits of the scaling relations for Monte Carlo simulations in diffuse optics. Part 1: theory.

Monte Carlo (MC) is a powerful tool to study photon migration in scattering media, yet quite time-consuming to solve inverse problems. To speed up MC-simulations, scaling relations can be applied to an existing initial MC-simulation to generate a new data-set with different optical properties. We named this approach trajectory-based since it uses the knowledge of the detected photon trajectories of the initial MC-simulation, in opposition to the slower photon-based approach, where a novel MC-simulation is rerun with new optical properties. We investigated the convergence and applicability limits of the scaling relations, both related to the likelihood that the sample of trajectories considered is representative also for the new optical properties. For absorption, the scaling relation contains smoothly converging Lambert-Beer factors, whereas for scattering it is the product of two quickly diverging factors, whose ratio, for NIRS cases, can easily reach ten orders of magnitude. We investigated such instability by studying the probability-distribution for the number of scattering events in trajectories of given length. We propose a convergence test of the scattering scaling relation based on the minimum-maximum number of scattering events in recorded trajectories. We also studied the dependence of MC-simulations on optical properties, most critical in inverse problems, finding that scattering derivatives are ascribed to small deviations in the distribution of scattering events from a Poisson distribution. This paper, which can also serve as a tutorial, helps to understand the physics of the scaling relations with the causes of their limitations and devise new strategies to deal with them.

Characterization of the Optical and Thermal Properties of Cardiac Tissue as a Function of Temperature.

In this work, we devised the first characterization of the optical and thermal properties of ex vivo cardiac tissue as a function of different selected temperatures, ranging from room temperature to hyperthermic and ablative temperatures. The broadband (i.e., from 650 nm to 1100 nm) estimation of the optical properties, i.e., absorption coefficient (µa) and reduced scattering coefficient $({\mu ^{\prime}}_s)$, was performed by means of time-domain diffuse optics. Besides, the measurement of the thermal properties was based on the transient hot-wire technique, employing a dual-needle probe to estimate the tissue thermal conductivity (k), thermal diffusivity (α), and volumetric heat capacity (Cv). Increasing the tissue temperature led to variations in the spectral characteristics of µa (e.g., the redshift of the 780 nm peak, the rise of a new peak at 840 nm, and the formation of a valley at 900 nm). Moreover, an increase in the values of ${\mu ^{\prime}}_s$ was assessed as tissue temperature raised (e.g., for 800 nm, at 25 °C ${\mu ^{\prime}}_s = 9.8{\text{ c}}{{\text{m}}^{{\text{ - 1}}}}$, while at 77 °C ${\mu ^{\prime}}_s = 29.1{\text{ c}}{{\text{m}}^{{\text{ - 1}}}}$). Concerning the thermal properties characterization, k was almost constant in the selected temperature interval. Conversely, α and Cv were subjected to an increase and a decrease with temperature, respectively; thus, they registered values of 0.190 mm2/s and 3.03 MJ/(m3•K) at the maximum investigated temperature (79 °C), accordingly.Clinical Relevance- The experimentally obtained optical and thermal properties of cardiac tissue are useful to improve the accuracy of simulation-based tools for thermal therapy planning. Furthermore, the measured properties can serve as a reference for the realization of tissue-mimicking phantoms for medical training and testing of medical instruments.

Two-layer reconstruction of Raman spectra in diffusive media based on an analytical model in the time domain.

We derive and validate an analytical model that describes the migration of Raman scattered photons in two-layer diffusive media, based on the diffusion equation in the time domain. The model is derived under a heuristic approximation that background optical properties are identical on the excitation and Raman emission wavelengths. Methods for the reconstruction of two-layer Raman spectra have been developed, tested in computer simulations and validated on tissue-mimicking phantom measurements data. Effects of different parameters were studied in simulations, showing that the thickness of the top layer and number of detected photon counts have the most significant impact on the reconstruction. The concept of quantitative, mathematically rigorous reconstruction using the proposed model was finally proven on experimental measurements, by successfully separating the spectra of silicone and calcium carbonate (calcite) layers, showing the potential for further development and eventual application in clinical diagnostics.

Time domain diffuse Raman spectroscopy using single pixel detection.

Diffuse Raman spectroscopy (DIRS) extends the high chemical specificity of Raman scattering to in-depth investigation of thick biological tissues. We present here a novel approach for time-domain diffuse Raman spectroscopy (TD-DIRS) based on a single-pixel detector and a digital micromirror device (DMD) within an imaging spectrometer for wavelength encoding. This overcomes the intrinsic complexity and high cost of detection arrays with ps-resolving time capability. Unlike spatially offset Raman spectroscopy (SORS) or frequency offset Raman spectroscopy (FORS), TD-DIRS exploits the time-of-flight distribution of photons to probe the depth of the Raman signal at a single wavelength with a single source-detector separation. We validated the system using a bilayer tissue-bone mimicking phantom composed of a 1 cm thick slab of silicone overlaying a calcium carbonate specimen and demonstrated a high differentiation of the two Raman signals. We reconstructed the Raman spectra of the two layers, offering the potential for improved and quantitative material analysis. Using a bilayer phantom made of porcine muscle and calcium carbonate, we proved that our system can retrieve Raman peaks even in the presence of autofluorescence typical of biomedical tissues. Overall, our novel TD-DIRS setup proposes a cost-effective and high-performance approach for in-depth Raman spectroscopy in diffusive media.

Fast time-domain diffuse correlation spectroscopy with superconducting nanowire single-photon detector: system validation and in vivo results.

Time-domain diffuse correlation spectroscopy (TD-DCS) has been introduced as an advancement of the "classical" continuous wave DCS (CW-DCS) allowing one to not only to measure depth-resolved blood flow index (BFI) but also to extract optical properties of the measured medium without using any additional diffuse optics technique. However, this method is a photon-starved technique, specially when considering only the late photons that are of primary interest which has limited its in vivo application. In this work, we present a TD-DCS system based on a superconducting nanowire single-photon detector (SNSPD) with a high quantum efficiency, a narrow timing response, and a negligibly low dark count noise. We compared it to the typically used single-photon avalanche diode (SPAD) detector. In addition, this system allowed us to conduct fast in vivo measurements and obtain gated pulsatile BFI on the adult human forehead.

Interstitial null-distance time-domain diffuse optical spectroscopy using a superconducting nanowire detector.

Significance: Interstitial fiber-based spectroscopy is gaining interest for real-time in vivo optical biopsies, endoscopic interventions, and local monitoring of therapy. Different from other photonics approaches, time-domain diffuse optical spectroscopy (TD-DOS) can probe the tissue at a few cm distance from the fiber tip and disentangle absorption from the scattering properties. Nevertheless, the signal detected at a short distance from the source is strongly dominated by the photons arriving early at the detector, thus hampering the possibility of resolving late photons, which are rich in information about depth and absorption. Aim: To fully benefit from the null-distance approach, a detector with an extremely high dynamic range is required to effectively collect the late photons; the goal of our paper is to test its feasibility to perform TD-DOS measurements at null source-detector separations (NSDS). Approach: In particular, we demonstrate the use of a superconducting nanowire single photon detector (SNSPD) to perform TD-DOS at almost NSDS ( ≈ 150 µ m ) by exploiting the high dynamic range and temporal resolution of the SNSPD to extract late arriving, deep-traveling photons from the burst of early photons. Results: This approach was demonstrated both on Monte Carlo simulations and on phantom measurements, achieving an accuracy in the retrieval of the water spectrum of better than 15%, spanning almost two decades of absorption change in the 700- to 1100-nm range. Additionally, we show that, for interstitial measurements at null source-detector distance, the scattering coefficient has a negligible effect on late photons, easing the retrieval of the absorption coefficient. Conclusions: Utilizing the SNSPD, broadband TD-DOS measurements were performed to successfully retrieve the absorption spectra of the liquid phantoms. Although the SNSPD has certain drawbacks for use in a clinical system, it is an emerging field with research progressing rapidly, and this makes the SNSPD a viable option and a good solution for future research in needle guided time-domain interstitial fiber spectroscopy.

Optical imaging and spectroscopy for the study of the human brain: status report.

This report is the second part of a comprehensive two-part series aimed at reviewing an extensive and diverse toolkit of novel methods to explore brain health and function. While the first report focused on neurophotonic tools mostly applicable to animal studies, here, we highlight optical spectroscopy and imaging methods relevant to noninvasive human brain studies. We outline current state-of-the-art technologies and software advances, explore the most recent impact of these technologies on neuroscience and clinical applications, identify the areas where innovation is needed, and provide an outlook for the future directions.

Estimation of porcine pancreas optical properties in the 600-1100 nm wavelength range for light-based therapies.

This work reports the optical properties of porcine pancreatic tissue in the broad wavelength range of 600-1100 nm. Absorption and reduced scattering coefficients (µa and µs') of the ex vivo pancreas were obtained by means of Time-domain Diffuse Optical Spectroscopy. We have investigated different experimental conditions-including compression, repositioning, spatial sampling, temporal stability-the effect of the freezing procedure (fresh vs frozen-thawed pancreas), and finally inter-sample variability. Good repeatability under different experimental conditions was obtained (median coefficient of variation less than 8% and ~ 16% for µa and µs', respectively). Freezing-thawing the samples caused an irreversible threefold reduction of µs' and no effect on µa. The absorption and reduced scattering spectra averaged over different samples were in the range of 0.12-0.74 cm-1 and 12-21 cm-1 with an inter-sample variation of ~ 10% and ~ 40% for µa and µs', respectively. The calculated effective transport coefficient (µeff) for fresh pancreatic tissue shows that regions between 800-900 nm and 1050-1100 nm are similar and offer the lowest tissue attenuation in the considered range (i.e., µeff ranging from 2.4 to 2.7 cm-1). These data, describing specific light-pancreas interactions in the therapeutic optical window for the first time, provide pivotal information for planning of light-based thermotherapies (e.g., laser ablation) and instruction of light transport models for biophotonic applications involving this organ.

Criteria for the design of tissue-mimicking phantoms for the standardization of biophotonic instrumentation.

A lack of accepted standards and standardized phantoms suitable for the technical validation of biophotonic instrumentation hinders the reliability and reproducibility of its experimental outputs. In this Perspective, we discuss general criteria for the design of tissue-mimicking biophotonic phantoms, and use these criteria and state-of-the-art developments to critically review the literature on phantom materials and on the fabrication of phantoms. By focusing on representative examples of standardization in diffuse optical imaging and spectroscopy, fluorescence-guided surgery and photoacoustic imaging, we identify unmet needs in the development of phantoms and a set of criteria (leveraging characterization, collaboration, communication and commitment) for the standardization of biophotonic instrumentation.

Evaluation of a pipeline for simulation, reconstruction, and classification in ultrasound-aided diffuse optical tomography of breast tumors.

SIGNIFICANCE: Diffuse optical tomography is an ill-posed problem. Combination with ultrasound can improve the results of diffuse optical tomography applied to the diagnosis of breast cancer and allow for classification of lesions. AIM: To provide a simulation pipeline for the assessment of reconstruction and classification methods for diffuse optical tomography with concurrent ultrasound information. APPROACH: A set of breast digital phantoms with benign and malignant lesions was simulated building on the software VICTRE. Acoustic and optical properties were assigned to the phantoms for the generation of B-mode images and optical data. A reconstruction algorithm based on a two-region nonlinear fitting and incorporating the ultrasound information was tested. Machine learning classification methods were applied to the reconstructed values to discriminate lesions into benign and malignant after reconstruction. RESULTS: The approach allowed us to generate realistic US and optical data and to test a two-region reconstruction method for a large number of realistic simulations. When information is extracted from ultrasound images, at least 75% of lesions are correctly classified. With ideal two-region separation, the accuracy is higher than 80%. CONCLUSIONS: A pipeline for the generation of realistic ultrasound and diffuse optics data was implemented. Machine learning methods applied to a optical reconstruction with a nonlinear optical model and morphological information permit to discriminate malignant lesions from benign ones.

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.

Time-domain NIRS system based on supercontinuum light source and multi-wavelength detection: validation for tissue oxygenation studies.

We present and validate a multi-wavelength time-domain near-infrared spectroscopy (TD-NIRS) system that avoids switching wavelengths and instead exploits the full capability of a supercontinuum light source by emitting and acquiring signals for the whole chosen range of wavelengths. The system was designed for muscle and brain oxygenation monitoring in a clinical environment. A pulsed supercontinuum laser emits broadband light and each of two detection modules acquires the distributions of times of flight of photons (DTOFs) for 16 spectral channels (used width 12.5 nm / channel), providing a total of 32 DTOFs at up to 3 Hz. Two emitting fibers and two detection fiber bundles allow simultaneous measurements at two positions on the tissue or at two source-detector separations. Three established protocols (BIP, MEDPHOT, and nEUROPt) were used to quantitatively assess the system's performance, including linearity, coupling, accuracy, and depth sensitivity. Measurements were performed on 32 homogeneous phantoms and two inhomogeneous phantoms (solid and liquid). Furthermore, measurements on two blood-lipid phantoms with a varied amount of blood and Intralipid provide the strongest validation for accurate tissue oximetry. The retrieved hemoglobin concentrations and oxygen saturation match well with the reference values that were obtained using a commercially available NIRS system (OxiplexTS) and a blood gas analyzer (ABL90 FLEX), except a discrepancy occurs for the lowest amount of Intralipid. In-vivo measurements on the forearm of three healthy volunteers during arterial (250 mmHg) and venous (60 mmHg) cuff occlusions provide an example of tissue monitoring during the expected hemodynamic changes that follow previously well-described physiologies. All results, including quantitative parameters, can be compared to other systems that report similar tests. Overall, the presented TD-NIRS system has an exemplary performance evaluated with state-of-the-art performance assessment methods.

Performance assessment of laser sources for time-domain diffuse correlation spectroscopy.

Time-domain diffuse correlation spectroscopy (TD-DCS) is an emerging optical technique that enables noninvasive measurement of microvascular blood flow with photon path-length resolution. In TD-DCS, a picosecond pulsed laser with a long coherence length, adequate illumination power, and narrow instrument response function (IRF) is required, and satisfying all these features is challenging. To this purpose, in this study we characterized the performance of three different laser sources for TD-DCS. First, the sources were evaluated based on their emission spectrum and IRF. Then, we compared the signal-to-noise ratio and the sensitivity to velocity changes of scattering particles in a series of phantom measurements. We also compared the results for in vivo measurements, performing an arterial occlusion protocol on the forearm of three adult subjects. Overall, each laser has the potential to be successfully used both for laboratory and clinical applications. However, we found that the effects caused by the IRF are more significant than the effect of a limited temporal coherence.

Beyond multi view deconvolution for inherently aligned fluorescence tomography.

In multi-view fluorescence microscopy, each angular acquisition needs to be aligned with care to obtain an optimal volumetric reconstruction. Here, instead, we propose a neat protocol based on auto-correlation inversion, that leads directly to the formation of inherently aligned tomographies. Our method generates sharp reconstructions, with the same accuracy reachable after sub-pixel alignment but with improved point-spread-function. The procedure can be performed simultaneously with deconvolution further increasing the reconstruction resolution.

Time resolved speckle contrast optical spectroscopy at quasi-null source-detector separation for non-invasive measurement of microvascular blood flow.

Time (or path length) resolved speckle contrast optical spectroscopy (TD-SCOS) at quasi-null (2.85 mm) source-detector separation was developed and demonstrated. The method was illustrated by in vivo studies on the forearm muscle of an adult subject. The results have shown that selecting longer photon path lengths results in higher hyperemic blood flow change and a faster return to baseline by a factor of two after arterial cuff occlusion when compared to SCOS without time resolution. This indicates higher sensitivity to the deeper muscle tissue. In the long run, this approach may allow the use of simpler and cheaper detector arrays compared to time resolved diffuse correlation spectroscopy that are based on readily available technologies. Hence, TD-SCOS may increase the performance and decrease cost of devices for continuous non-invasive, deep tissue blood flow monitoring.

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.