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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.

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.

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.

SIGNIFICANCE: Multi-laboratory initiatives are essential in performance assessment and standardization-crucial for bringing biophotonics to mature clinical use-to establish protocols and develop reference tissue phantoms that all will allow universal instrument comparison. AIM: The largest multi-laboratory comparison of performance assessment in near-infrared diffuse optics is presented, involving 28 instruments and 12 institutions on a total of eight experiments based on three consolidated protocols (BIP, MEDPHOT, and NEUROPT) as implemented on three kits of tissue phantoms. A total of 20 synthetic indicators were extracted from the dataset, some of them defined here anew. APPROACH: The exercise stems from the Innovative Training Network BitMap funded by the European Commission and expanded to include other European laboratories. A large variety of diffuse optics instruments were considered, based on different approaches (time domain/frequency domain/continuous wave), at various stages of maturity and designed for different applications (e.g., oximetry, spectroscopy, and imaging). RESULTS: This study highlights a substantial difference in hardware performances (e.g., nine decades in responsivity, four decades in dark count rate, and one decade in temporal resolution). Agreement in the estimates of homogeneous optical properties was within 12% of the median value for half of the systems, with a temporal stability of

Diffuse optics studies the propagation of light in highly scattering media (e.g. biological tissues). Time-resolved techniques – which measure the time-of-flight distribution of photons in the medium – permit to separate the contribution of absorption (linked to chemical composition) from scattering (produced by microstructure). Furthermore, for increasing photon time-of-flight, larger tissue depths are reached. These two properties, combined with the intrinsic non-invasiveness of the technique, are very interesting for clinical applications, such as diagnosis of breast cancer, imaging of brain activity, monitoring the functional and metabolic state of biological tissues in depth. The technology has undergone impressive growth, moving from laboratory laser systems to miniature wearable devices. To grant the reliability of the information obtained in vivo, international collaboration is needed to set objective validation protocols, in view of a growing diffusion of the method, from clinical diagnostics to home care.

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.

The original version of this Article contained errors in references 3, 4, 25, 36, 41 and 48, which were incorrectly given as

The ability to non-invasively monitor in-vivo the human muscle and adipose tissue is of great practical use and hence of growing interest in the fields of clinical diagnostics and preventive medicine. Optical methods, such as diffuse optical spectroscopy (DOS) applied in the near-infrared spectral region could be of great interest in clinical scenario. In this work, we present a pilot study based on multi-distance broadband time-domain diffuse optical spectroscopy (TD DOS) to characterize in vivo the subcutaneous adipose tissue (abdominal region) and the vastus lateralis muscle (thigh region). The study was performed using a fully automated portable TD DOS instrument on a set of 24 healthy adult volunteers. The optical properties of these two tissue types were obtained over the broad wavelength range of 600-1100 nm. The results suggest a clear influence of the stratified nature of the two regions considered, namely the abdomen and thigh, on the recovered optical properties. This work demonstrates how multi-distance broadband diffuse optical spectroscopy could be complimentary in fields like the non-invasive spectroscopy of adipose tissue and the standard DOS-based muscle oximetry.

We have addressed the challenge of investigating the lung using diffuse optics along four different directions, namely: 1) broadband time-domain diffuse optics (TD-DO) in the 600-1100 nm range to derive the mean chest optical properties; 2) Monte Carlo simulations to investigate the depth-sensitivity of TD-DO measurements assuming a layered structure of the chest; 3) single-wavelength TD-DO using a high power pulsed laser on 5 healthy volunteers on a dynamic protocol; 4) single-wavelength TD-DO measurements using a novel large area Silicon Photomultiplier (SIPM) detector module permitting acquisitions at 12 cm source-detector distance.

Thanks to recent developments in time-domain near-infrared spectroscopy (TD-NIRS), it is now possible to monitor brain activity during freely moving exercises. A wearable single-channel TD-NIRS instrument has been exploited to retrieve oxy- and deoxygenated hemoglobin absolute concentrations during gaiting experiments. It was possible to retrieve quantitative results concerning functional brain activation in the motor cortex areas, with high rejection of motion artifacts.

A standardized approach to develop a reliable, reproducible, stable phantoms was proposed. A well-established instrument validation protocol (MEDPHOT) was adopted for this purpose. This approach was tested on two phantom recipes (silicone and polyurethane) over broadband (600-1100 nm) wavelength covering a wider range of optical properties (absorption 0.1-1 cm-1, reduced scattering 5-20 cm-1) relevant to human tissue. As an application of the recipe, a reliable tissue-mimicking 3D anthropomorphic head phantom was presented.

Time-domain diffuse optics exploits near infrared light pulses diffused in turbid samples to retrieve their optical properties e.g., absorption and reduced scattering coefficients. Typically, interference effect are discarded, but speckle effects are exploited in other techniques e.g., diffuse correlation spectroscopy (DCS) to retrieve information regarding the tissue dynamics. Here, using a highly coherent Ti:Sapphire mode-locked laser and a single-mode detection fiber, we report the direct observation of temporal fluctuations in the measured distribution of time-of-flights (DTOF) curve. We study the dependence of these fluctuations on the sample dynamical properties (moving from fluid to rigid tissue-mimicking phantoms) and on the area of the detection fiber, which is directly linked to the number of collected coherence areas. Our observation agree with a time-resolved speckle pattern, and may enable the simultaneous monitoring of the tissue optical and dynamical properties.

Diffuse correlation spectroscopy (DCS) is an optical technique which, by studying the speckle intensity fluctuations of coherent light diffused in a turbid medium, retrieves information regarding the scatterers motion. In the case of biological tissues, the particles of interest are the red blood cells, from which is possible to measure non-invasively microvascular blood flow (BF). However, being based on a continuous-wave light source, depth discrimination is achievable only by using multiple source-detector separations. On the other hand, time-domain (TD) DCS is a novel approach which exploits a pulsed yet coherent light source to discriminate the intensity fluctuations at different photon time-of-flights. This additional information is beneficial for in vivo applications, due to the physical relationship between photon time-of-flight and mean depth penetration. TD-DCS is typically performed in the spectral range between 700 and 800 nm. Here, we explore TD-DCS in a new spectral range compared to the typical one, moving to the spectral region beyond the water absorption peak (i.e., >970 nm). We performed liquid phantom and in vivo experiments on the human muscle at a wavelength of 1000 nm. Also, the possible advantages in terms of depth sensitivity and signal-to-noise ratio are discussed.

The use of technologies still under development (Silicon PhotoMultipliers and Time-to-Digital Converter) for the upgrade of our time-resolved optical mammograph highlighted the crucial need for a thorough and progressive instrument characterization (from extensive laboratory tests on phantoms to in vivo measurements), potentially of general interest. Despite satisfying results on phantoms, in vivo tests urged an instrumental change, with the adoption of a just released high-throughput TDC to obtain high-quality breast scans (much wider scan area, better day-by-day reproducibility, improved signal quality, simpler set-up). A clinical study on monitoring of neoadjuvant chemotherapy is now ongoing at the San Raffaele Hospital, Milan.

Performance assessment and standardization are indispensable for instruments of clinical relevance in general and clinical instrumentation based on photon migration/diffuse optics in particular. In this direction, a multi-laboratory exercise was initiated with the aim of assessing and comparing their performances. 29 diffuse optical instruments belonging to 11 partner institutions of a European level Marie Curie Consortium BitMap1 were considered for this exercise. The enrolled instruments covered different approaches (continuous wave, CW; frequency domain, FD; time domain, TD and spatial frequency domain imaging, SFDI) and applications (e.g. mammography, oximetry, functional imaging, tissue spectroscopy). 10 different tests from 3 well-accepted protocols, namely, the MEDPHOT2 , the BIP3 , and the nEUROPt4 protocols were chosen for the exercise and the necessary phantoms kits were circulated across labs and institutions enrolled in the study. A brief outline of the methodology of the exercise is presented here. Mainly, the design of some of the synthetic descriptors, (single numeric values used to summarize the result of a test and facilitate comparison between instruments) for some of the tests will be discussed.. Future actions of the exercise aim at deploying these measurements onto an open data repository and investigating common analysis tools for the whole dataset.

To improve non-invasively the specificity in the diagnosis of breast cancer after a positive screening mammography or doubt/suspicious ultrasound examination, the SOLUS project developed a multimodal imaging system that combines: Bmode ultrasound (US) scans (to assess morphology), Color Doppler (to visualize vascularization), shear-wave elastography (to measure stiffness), and time domain multi-wavelength diffuse optical tomography (to estimate tissue composition in terms of oxy- and deoxy-hemoglobin, lipid, water, and collagen concentrations). The multimodal probe arranges 8 innovative photonic modules (optodes) around the US transducer, providing capability for optical tomographic reconstruction. For more accurate estimate of lesion composition, US-assessed morphological priors can be used to guide the optical reconstructions. Each optode comprises: i) 8 picosecond pulsed laser diodes with different wavelengths, covering a wide spectral range (635-1064 nm) for good probing of the different tissue constituents; ii) a large-area (variable, up to 8.6 mm2 ) fast-gated digital Silicon Photomultiplier; iii) the acquisition electronics to record the distribution of time-of-flight of the re-emitted photons. The optode is the basic element of the optical part of the system, but is also a stand-alone, ultra-compact (about 4 cm3 ) device for time domain multi-wavelength diffuse optics, with potential application in various fields.

Multimodal imaging is an active branch of research as it has the potential to improve common medical imaging techniques. Diffuse optical tomography (DOT) is an example of a low resolution, functional imaging modality that typically has very low resolution due to the ill-posedness of its underlying inverse problem. Combining the functional information of DOT with a high resolution structural imaging modality has been studied widely. In particular, the combination of DOT with ultrasound (US) could serve as a useful tool for clinicians for the formulation of accurate diagnosis of breast lesions. In this paper, we propose a novel method for US-guided DOT reconstruction using a portable time-domain measurement system. B-mode US imaging is used to retrieve morphological information on the probed tissues by means of a semi-automatical segmentation procedure based on active contour fitting. A two-dimensional to three-dimensional extrapolation procedure, based on the concept of distance transform, is then applied to generate a three-dimensional edge-weighting prior for the regularization of DOT. The reconstruction procedure has been tested on experimental data obtained on specifically designed dual-modality silicon phantoms. Results show a substantial quantification improvement upon the application of the implemented technique. This article is part of the theme issue ‘Synergistic tomographic image reconstruction: part 2’.

Tomographic inspection of fluorescent labels distributed within a specimen is an important aspect in biology. Light sheet fluorescent microscopy (LSFM) offers a powerful and simple tool to selectively slice the sample and let us directly obtain a tomographic view of the specimen. However, due to non-isotropic resolution of the technique along the axial scanning, one may want to combine different views of the object and add deconvolution to the process in order to achieve higher resolution. Typically, multi-view Bayesian methods based on Richardson-Lucy deconvolution are used for this task once the datasets are exactly registered against each other. In this work, instead, we begin to investigate how to avoid the alignment procedure and use a direct algorithm to form a multi-view tomographic reconstruction. To do this, we developed a new framework based on auto-correlation analysis that let us achieve deconvolved reconstructions starting from blurred auto-correlations. Since the latter are insensitive to shifts, we can combine the auto-correlations coming from multi-view acquisitions without taking care of the registration procedure.

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.

Bulkiness and complexity of current Time-Domain (TD) NIRS systems have strongly limited the spread of this technique in biomedical research and clinical applications. We present an instrument developed to overcome those limitations, making TD-NIRS more accessible to non-experts and adaptable to any measurement condition, paving the way to muscle and brain oximetry in healthcare and sport environments.

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.