Significance: Pharmacokinetic parametric images in dynamic fluorescence molecular tomography (FMT) can describe three-dimensional (3D) physiological and pathological information inside biological tissues, potentially providing quantitative assessment tools for biological research and drug development.

Aim: In vivo imaging of the liver tumor with pharmacokinetic parametric images from dynamic FMT based on the differences in metabolic properties of indocyanine green (ICG) between normal liver cells and tumor liver cells inside biological tissues.

Approach: First, an orthotopic liver tumor mouse model was constructed. Then, with the help of the FMT/computer tomography (CT) dual-modality imaging system and the direct reconstruction algorithm, 3D imaging of liver metabolic parameters in nude mice was achieved to distinguish liver tumors from normal tissues. Finally, pharmacokinetic parametric imaging results were validated against in vitro anatomical results.

Results: This letter demonstrates the ability of dynamic FMT to monitor the pharmacokinetic delivery of the fluorescent dye ICG in vivo, thus, enabling the distinction between normal and tumor tissues based on the pharmacokinetic parametric images derived from dynamic FMT.

Conclusions: Compared with CT structural imaging technology, dynamic FMT combined with compartmental modeling as an analytical method can obtain quantitative images of pharmacokinetic parameters, thus providing a more powerful research tool for organ function assessment, disease diagnosis and new drug development.

Significance: Exogenous extracellular matrix (ECM) proteins, such as fibrinogen and the thrombin-polymerized scaffold fibrin, are used in surgical repair of severe nerve injuries to supplement ECM produced via the injury response. Monitoring the dynamic changes of fibrin during nerve regeneration may shed light on the frequent failure of grafts in the repair of long nerve gaps.

Aim: We explored whether monitoring of fibrin dynamics can be carried out using nerve guidance conduits (NGCs) containing fibrin tagged with covalently bound fluorophores.

Approach: Fibrinogen was conjugated to a near-infrared (NIR) fluorescent dye. NGCs consisting of silicone tubes filled with the fluorescent fibrin were used to repair a 5-mm gap injury in rat sciatic nerve (n = 6).

Results: Axonal regeneration in fluorescent fibrin-filled NGCs was confirmed at 14 days after implantation. Intraoperative fluorescence imaging after implantation showed that the exogenous fibrin was embedded in the early stage regenerative tissue. The fluorescent signal temporarily highlighted a cable-like structure within the conduit and gradually degraded over two weeks.

Conclusions: This study, for the first time, visualized in vivo intraneural fibrin degradation, potentially a useful prospective indicator of regeneration success, and showed that fluorescent ECM, in this case fibrin, can facilitate imaging of regeneration in peripheral nerve conduits without significantly affecting the regeneration process.

Significance: Deep tissue noninvasive high-resolution imaging with light is challenging due to the high degree of light absorption and scattering in biological tissue. Photoacoustic imaging (PAI) can overcome some of the challenges of pure optical or ultrasound imaging to provide high-resolution deep tissue imaging. However, label-free PAI signals from light absorbing chromophores within the tissue are nonspecific. The use of exogeneous contrast agents (probes) not only enhances the imaging contrast (and imaging depth) but also increases the specificity of PAI by binding only to targeted molecules and often providing signals distinct from the background.

Aim: We aim to review the current development and future progression of photoacoustic molecular probes/contrast agents.

Approach: First, PAI and the need for using contrast agents are briefly introduced. Then, the recent development of contrast agents in terms of materials used to construct them is discussed. Then, various probes are discussed based on targeting mechanisms, in vivo molecular imaging applications, multimodal uses, and use in theranostic applications.

Results: Material combinations are being used to develop highly specific contrast agents. In addition to passive accumulation, probes utilizing activation mechanisms show promise for greater controllability. Several probes also enable concurrent multimodal use with fluorescence, ultrasound, Raman, magnetic resonance imaging, and computed tomography. Finally, targeted probes are also shown to aid localized and molecularly specific photo-induced therapy.

Conclusions: The development of contrast agents provides a promising prospect for increased contrast, higher imaging depth, and molecularly specific information. Of note are agents that allow for controlled activation, explore other optical windows, and enable multimodal use to overcome some of the shortcomings of label-free PAI.

Significance: Reflectance confocal microscopy (RCM) is a noninvasive, in vivo technology that offers near histopathological resolution at the cellular level. It is useful in the study of phenomena for which obtaining a biopsy is impractical or would cause unnecessary tissue damage and trauma to the patient.

Aim: This review covers the use of RCM in the study of skin and the use of machine learning to automate information extraction. It has two goals: (1) an overview of information provided by RCM on skin structure and how it changes over time in response to stimuli and in disease and (2) an overview of machine learning approaches developed to automate the extraction of key morphological features from RCM images.

Approach: A PubMed search was conducted with additional literature obtained from references lists.

Results: The application of RCM as an in vivo tool in dermatological research and the biologically relevant information derived from it are presented. Algorithms for image classification to epidermal layers, delineation of the dermal–epidermal junction, classification of skin lesions, and demarcation of individual cells within an image, all important factors in the makeup of the skin barrier, were reviewed. Application of image analysis methods in RCM is hindered by low image quality due to noise and/or poor contrast. Use of supervised machine learning is limited by time-consuming manual labeling of RCM images.

Conclusions: RCM has great potential in the study of skin structures. The use of artificial intelligence could enable an easier, more reproducible, precise, and rigorous study of RCM images for the understanding of skin structures, skin barrier, and skin inflammation and lesions. Although several attempts have been made, further work is still needed to provide a definite gold standard and overcome issues related to image quality, limited labeled datasets, and lack of phenotype variability in available databases.

The editorial introduces the JBO Special Section on Tissue Phantoms to Advance Biomedical Optical Systems.

Significance: Fluorescence imaging of rheumatoid diseases with indocyanine green (ICG) is an emerging technique with unique potential for diagnosis and therapy. Device characterization, monitoring of the performance, and further developments of the technique require tissue-equivalent fluorescent phantoms of high stability with appropriate anatomical shapes.

Aim: Our investigations aim at the development of a three-dimensional (3D) printing technique to fabricate hand and finger models with appropriate optical properties in the near-infrared spectral range. These phantoms should have fluorescence properties similar to ICG, and excellent photostability and durability over years.

Approach: We modified a 3D printing methacrylate photopolymer by adding the fluorescent dye Lumogen IR 765 to the raw material. Reduced scattering and absorption coefficients were adjusted to values representative of the human hand by incorporating titanium dioxide powder and black ink. The properties of printed phantoms of various compositions were characterized using UV/Vis and fluorescence spectroscopy, and time-resolved measurements. Photostability and bleaching were investigated with a hand imager. For comparison, several phantoms with ICG as fluorescent dye were printed and characterized as well.

Results: The spectral properties of Lumogen IR 765 are very similar to those of ICG. By optimizing the concentrations of Lumogen, titanium dioxide, and ink, anatomically shaped hand and vessel models with properties equivalent to in vivo investigations with a fluorescence hand imager could be printed. Phantoms with Lumogen IR 765 had an excellent photostability over up to 4 years. In contrast, phantoms printed with ICG showed significant bleaching and degradation of fluorescence over time.

Conclusions: 3D printing of phantoms with Lumogen IR 765 is a promising method for fabricating anatomically shaped fluorescent tissue models of excellent stability with spectral properties similar to ICG. The phantoms are well-suited to monitor the performance of hand imagers. Concepts can easily be transferred to other fluorescence imaging applications of ICG.

Significance: Cerebral tissue oximetry is imprecise and confounded by an uncertain and variable arteriovenous volume ratio. Venous saturation is better grounded in physiology. The superior sagittal sinus (SSS) is relatively large and placed under the open fontanel on the top of the head in newborn infants.

Aim: To enable the development of a dedicated near-infrared-spectroscopy-based cerebral oximeter with sufficient claims on accuracy to be tested for benefit of clinical use.

Approach: To set up a research agenda based on the combination of dedicated, high-fidelity digital and physical phantoms.

Results: A seven-step path is outlined to identify an optode geometry with high sensitivity to variation in hemoglobin-oxygen saturation in the SSS, with little confounding by changes in the optical properties of the skin and scalp or brain tissue, or in the width of the subarachnoidal space, and that is robust to variations in the placement of the optode.

Conclusion: If an oximeter that is designed after exploration of digital phantoms can produce measurements in physical phantoms with good agreement with predictions, it will contribute credibility that cannot be achieved by direct gold-standard validation in newborn human infants.

Significance: Near-infrared fluorescence molecular endoscopy (NIR-FME) is an innovative technique allowing for in vivo visualization of molecular processes in hollow organs. Despite its potential for clinical translation, NIR-FME still faces challenges, for example, the lack of consensus in performing quality control and standardization of procedures and systems. This may hamper the clinical approval of the technology by authorities and its acceptance by endoscopists. Until now, several clinical trials using NIR-FME have been performed. However, most of these trials had different study designs, making comparison difficult.

Aim: We describe the need for standardization in NIR-FME, provide a pathway for setting up a standardized clinical study, and describe future perspectives for NIR-FME.

Body: Standardization is challenging due to many parameters. Invariable parameters refer to the hardware specifications. Variable parameters refer to movement or tissue optical properties. Phantoms can be of aid when defining the influence of these variables or when standardizing a procedure.

Conclusion: There is a need for standardization in NIR-FME and hurdles still need to be overcome before a widespread clinical implementation of NIR-FME can be realized. When these hurdles are overcome, clinical outcomes can be compared and systems can be benchmarked, enabling clinical implementation.

Significance: Raman spectroscopy has emerged as a promising technique for a variety of biomedical applications. The unique ability to provide molecular specific information offers insight to the underlying biochemical changes that result in disease states such as cancer. However, one of the hurdles to successful clinical translation is a lack of international standards for calibration and performance assessment of modern Raman systems used to interrogate biological tissue.

Aim: To facilitate progress in the clinical translation of Raman-based devices and assist the scientific community in reaching a consensus regarding best practices for performance testing.

Approach: We reviewed the current literature and available standards documents to identify methods commonly used for bench testing of Raman devices (e.g., relative intensity correction, wavenumber calibration, noise, resolution, and sensitivity). Additionally, a novel 3D-printed turbid phantom was used to assess depth sensitivity. These approaches were implemented on three fiberoptic-probe-based Raman systems with different technical specifications.

Results: While traditional approaches demonstrated fundamental differences due to detectors, spectrometers, and data processing routines, results from the turbid phantom illustrated the impact of illumination-collection geometry on measurement quality.

Conclusions: Specifications alone are necessary but not sufficient to predict in vivo performance, highlighting the need for phantom-based test methods in the standardized evaluation of Raman devices.

Significance: The polymer, polydimethylsiloxane (PDMS), has been increasingly used to make tissue simulating phantoms due to its excellent processability, durability, flexibility, and limited tunability of optical, mechanical, and thermal properties. We report on a robust technique to fabricate PDMS-based tissue-mimicking phantoms where the broad range of scattering and absorption properties are independently adjustable in the visible- to near-infrared wavelength range from 500 to 850 nm. We also report on an analysis method to concisely quantify the phantoms’ broadband characteristics with four parameters.

Aim: We report on techniques to manufacture and characterize solid tissue-mimicking phantoms of PDMS polymers. Tunability of the absorption (μ_a  (  λ  )  ) and reduced scattering coefficient spectra (μs′(λ)) in the wavelength range of 500 to 850 nm is demonstrated by adjusting the concentrations of light absorbing carbon black powder (CBP) and light scattering titanium dioxide powder (TDP) added into the PDMS base material.

Approach: The μ_a  (  λ  )   and μs′(λ) of the phantoms were obtained through measurements with a broadband integrating sphere system and by applying an inverse adding doubling algorithm. Analyses of μ_a  (  λ  )   and μs′(λ) of the phantoms, by fitting them to linear and power law functions, respectively, demonstrate that independent control of μ_a  (  λ  )   and μs′(λ) is possible by systematically varying the concentrations of CBP and TDP.

Results: Our technique quantifies the phantoms with four simple fitting parameters enabling a concise tabulation of their broadband optical properties as well as comparisons to the optical properties of biological tissues. We demonstrate that, to a limited extent, the scattering properties of our phantoms mimic those of human tissues of various types. A possible way to overcome this limitation is demonstrated with phantoms that incorporate polystyrene microbead scatterers.

Conclusions: Our manufacturing and analysis techniques may further promote the application of PDMS-based tissue-mimicking phantoms and may enable robust quality control and quality checks of the phantoms.

Significance: Gas in scattering media absorption spectroscopy (GASMAS) enables noninvasive gas sensing in the body. It is developing as a tool for diagnosis and monitoring of respiratory conditions in neonates. Phantom models with relevant features to the clinical translation of GASMAS technology are necessary to understand technical challenges and potential applications of this technique. State-of-the-art phantoms designed for this purpose have focused on the optical properties and anthropomorphic geometry of the thorax, contributing to the source–detector placement, design, and optimization. Lung phantom mimicking the alveolar anatomy has not been included in the existent models due to the inherent complexity of the tissue. We present a simplified model that recreates inflated alveoli embedded in lung phantom.

Aim: The goal of this study was to build a lung model with air-filled structures mimicking inflated alveoli surrounded by optical phantom with accurate optical properties (μ_a  =  0.50  cm^−  1 and μs′=5.4  cm−1) and physiological parameters [37°C and 100% relative humidity (RH)], and to control the air volume within the phantom to demonstrate the feasibility of GASMAS in sensing changes in pulmonary air volume.

Approach: The lung model was built using a capillary structure with analogous size to alveolar units. Part of the capillaries were filled with liquid lung optical phantom to recreate scattering and absorption, whereas empty capillaries mimicked air filled alveoli. The capillary array was placed inside a custom-made chamber that maintained pulmonary temperature and RH. The geometry of the chamber permitted the placement of the laser head and detector of a GASMAS bench top system (MicroLab Dual O₂  /  H₂O), to test the changes in volume of the lung model in transmittance geometry.

Results: The lung tissue model with air volume range from 6.89  ×  10^−  7  m³ to 1.80  ×  10^−  3  m³ was built. Two measurement sets, with 10 different capillary configurations each, were arranged to increase or decrease progressively (in steps of 3.93  ×  10^−  8  m³) the air volume in the lung model. The respective GASMAS data acquisition was performed for both data sets. The maximum absorption signal was obtained for configurations with the highest number of air-filled capillaries and decreased progressively when the air spaces were replaced by capillaries filled with liquid optical phantom. Further studies are necessary to define the minimum and maximum volume of air that can be measured with GASMAS-based devices for different source–detector geometries.

Conclusions: The optical properties and the structure of tissue from the respiratory zone have been modeled using a simplified capillary array immersed in a controlled environment chamber at pulmonary temperature and RH. The feasibility of measuring volume changes with GASMAS technique has been proven, stating a new possible application of GASMAS technology in respiratory treatment and diagnostics.

Significance: For optical methods to accurately assess hemoglobin oxygen saturation in vivo, an independently verifiable tissue-like standard is required for validation. For this purpose, we propose three hemoglobin preparations and evaluate methods to characterize them.

Aim: To spectrally characterize three different hemoglobin preparations using multiple spectroscopic methods and to compare their absorption spectra to commonly used reference spectra.

Approach: Absorption spectra of three hemoglobin preparations in solution were characterized using spectroscopic collimated transmission: whole blood, lysed blood, and ferrous-stabilized hemoglobin. Tissue-mimicking phantoms composed of Intralipid, and the hemoglobin solutions were characterized using spatial frequency-domain spectroscopy (SFDS) and enhanced perfusion and oxygen saturation (EPOS) techniques while using yeast to deplete oxygen.

Results: All hemoglobin preparations exhibited similar absorption spectra when accounting for methemoglobin and scattering in their oxyhemoglobin and deoxyhemoglobin forms, respectively. However, systematic differences were observed in the fitting depending on the reference spectra used. For the tissue-mimicking phantoms, SFDS measurements at the surface of the phantom were affected by oxygen diffusion at the interface with air, associated with higher values than for the EPOS system.

Conclusions: We show the validity of different blood phantoms and what considerations need to be addressed in each case to utilize them equivalently.

Significance: During the development and early testing phases of new photoacoustic (PA) breast imaging systems, several choices need to be made in aspects of system design and measurement sequences. Decision-making can be complex for state-of-the-art systems such as 3D hybrid photoacoustic-ultrasound (PA-US) breast imagers intended for multispectral quantitative imaging. These systems have a large set of design choices and system settings that affect imaging performance in different ways and often require trade-offs. Decisions have to be made carefully as they can strongly influence the imaging performance.

Aim: A systematic approach to assess the influence of various choices on the imaging performance in carefully controlled laboratory situations is crucial before starting with human studies. Test objects and phantoms are used for first imaging studies, but most reported structures have a 2D geometry and are not suitable to assess all the image quality characteristics (IQCs) of 3D hybrid PA-US systems.

Approach: Our work introduces a suite of five test objects designed for hybrid PA-US systems with a 3D detection aperture. We present the test object designs and production protocols and explain how they can be used to study various performance measures. To demonstrate the utility of the developed objects, measurements are made with an existing tomographic PA system.

Results: Two test objects were developed for measurements of the US detectors’ impulse responses and light distribution on the breast surface. Three others were developed to assess image quality and quantitative accuracy of the PA and US modes. Three of the five objects were imaged to demonstrate their use.

Conclusions: The developed test objects allow one to study influences of various choices in design and system settings. With this, IQCs can be assessed as a function of measurement sequence settings for the PA and US modes in a controlled way. Systematic studies and measurements using these objects will help to optimize various system settings and measurement protocols in laboratory situations before embarking on human studies.

Significance: Time-domain functional near-infrared spectroscopy (TD-fNIRS) has been considered as the gold standard of noninvasive optical brain imaging devices. However, due to the high cost, complexity, and large form factor, it has not been as widely adopted as continuous wave NIRS systems.

Aim: Kernel Flow is a TD-fNIRS system that has been designed to break through these limitations by maintaining the performance of a research grade TD-fNIRS system while integrating all of the components into a small modular device.

Approach: The Kernel Flow modules are built around miniaturized laser drivers, custom integrated circuits, and specialized detectors. The modules can be assembled into a system with dense channel coverage over the entire head.

Results: We show performance similar to benchtop systems with our miniaturized device as characterized by standardized tissue and optical phantom protocols for TD-fNIRS and human neuroscience results.

Conclusions: The miniaturized design of the Kernel Flow system allows for broader applications of TD-fNIRS.

Significance: Tissue birefringence is an important parameter to consider when designing realistic, tissue-mimicking phantoms. Options for suitable birefringent materials that can be used to accurately represent tissue scattering are limited.

Aim: To introduce a method of fabricating birefringent tissue phantoms with a commonly used material—polydimethylsiloxane (PDMS)—for imaging with polarization-sensitive optical coherence tomography (PS-OCT).

Approach: Stretch-induced birefringence was characterized in PDMS phantoms made with varying curing ratios, and the resulting phantom birefringence values were compared with those of biological tissues.

Results: We showed that, with induced birefringence levels up to 2.1  ×  10^−  4, PDMS can be used to resemble the birefringence levels in weakly birefringent tissues. We demonstrated the use of PDMS in the development of phantoms to mimic the normal and diseased bladder wall layers, which can be differentiated by their birefringence levels.

Conclusions: PDMS allows accurate control of tissue scattering and thickness, and it exhibits controllable birefringent properties. The use of PDMS as a birefringent phantom material can be extended to other birefringence imaging systems beyond PS-OCT and to mimic other organs.

Significance: Tissue simulating phantoms are an important part of validating biomedical optical techniques. Tissue pathology in inflammation and oedema involves changes in both water and hemoglobin fractions.

Aim: We present a method to create solid gelatin-based phantoms mimicking inflammation and oedema with adjustable water and hemoglobin fractions.

Approach: One store-bought gelatin and one research grade gelatin were evaluated. Different water fractions were obtained by varying the water-to-gelatin ratio. Ferrous stabilized human hemoglobin or whole human blood was added as absorbers, and the stability and characteristics of each were compared. Intralipid^® was used as the scatterer. All phantoms were characterized using spatial frequency domain spectroscopy.

Results: The estimated water fraction varied linearly with expected values (R²  =  0.96 for the store-bought gelatin and R²  =  0.99 for the research grade gelatin). Phantoms including ferrous stabilized hemoglobin stayed stable up to one day but had methemoglobin present at day 0. The phantoms with whole blood remained stable up to 3 days using the store-bought gelatin.

Conclusions: A range of physiological relevant water fractions was obtained for both gelatin types, with the stability of the phantoms including hemoglobin differing between the gelatin type and hemoglobin preparation. These low-cost phantoms can incorporate other water-based chromophores and be fabricated as thin sheets to form multilayered structures.

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.

Significance: The combination of reflectance and fluorescence spectroscopy allows the determination of tissue optical properties and the calculation of the intrinsic fluorescence in vivo. These parameters can discriminate between tissues and may allow the discrimination of malignant from benign tissue. While this approach has significant clinical potential, the lack of standardization and quality assessment prevents the upscaling of research.

Aim: Investigate which factors influence device calibration and tissue optical property determination. Improve system quality assessment and allow upscaling of the clinical research using multidiameter single fiber reflectance/singe fiber fluorescence spectroscopy.

Approach: Two studies, one phantom based on uniform calibrations and skin measurements and a clinical study including clinical calibrations. The first validates the effect of factors under identical conditions and the effect of calibration quality on the optical property determination of skin. The second shows the effect of different system configurations and the performance of the system and probe over an extended period.

Results: Phantom calibrations showed stability over a period of 20 weeks except for a 16-week-old intralipid phantom which showed a significant difference (at least p  =  0.0032) for all five probes evaluated. For clinical calibrations, only the fiber tree had a significant influence (probe 4: p  <  0.000001 and probe 5: p  =  0.00038) on the calibration quality. Interestingly, no degradation of probe performance was detected over a period of 21 months despite the exposure to stress during clinical measurements. Calibration quality affected μs′ and the power law scattering exponent, but the degree of the influence was different per fiber.

Conclusions: Intralipid phantom quality and fiber tree performance are the main factors influencing the calibration quality. Probe and user performance did not show any effect, which makes the upscaling of research to multicenter trials easier. A high-quality assessment procedure should be implemented to track changes during clinical trials.

Significance: The fluorescence-guided imaging for surgical intervention community recognizes the need for performance standards for these imaging devices. Tissue phantoms are used to track an imager’s performance as a fluorescence detector, but imager-specific units are of limited utility.

Aim: Tissue phantoms can be calibrated to be traceable to the international system of units (SI) and in turn be used to calibrate imagers such that fluorescence measurements can be reported in universally accepted units.

Approach: The radiometry to convert imager-specific arbitrary digital counts to SI-traceable unit of watts is described in this paper.

Results: An example of an imager calibration is included.

Conclusions: Calibrated tissue phantoms become a tool for metrological traceability.

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 <5  %   over 1 h, and day-to-day reproducibility of <3  %  . Other tests encompassed linearity, crosstalk, uncertainty, and detection of optical inhomogeneities.

Conclusions: This extensive multi-laboratory exercise provides a detailed assessment of near-infrared Diffuse optical instruments and can be used for reference grading. The dataset—available soon in an open data repository—can be evaluated in multiple ways, for instance, to compare different analysis tools or study the impact of hardware implementations.

Significance: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths with a best median survival of only 40 to 50 months for localized disease despite multimodal treatment. The standard tissue differentiation method continues to be pathology with histological staining analysis. Microscopic discrimination between inflammatory pancreatitis and malignancies is demanding.

Aim: We aim to accurately distinguish native pancreatic tissue using infrared (IR) spectroscopy in a fast and label-free manner.

Approach: Twenty cryopreserved human pancreatic tissue samples were collected from surgical resections. In total, more than 980,000 IR spectra were collected and analyzed using a MATLAB package. For differentiation of PDAC, pancreatitis, and normal tissue, a three-class training set for supervised classification was created with 25,000 spectra and the principal component analysis (PCA) score values for each cohort. Cross-validation was performed using the leave-one-out method. Validation of the algorithm was accomplished with 13 independent test samples.

Results: Reclassification of the training set and the independent test samples revealed an overall accuracy of more than 90% using a discrimination algorithm.

Conclusion: IR spectroscopy in combination with PCA and supervised classification is an efficient analytical method to reliably distinguish between benign and malignant pancreatic tissues. It opens up a wide research field for oncological and surgical applications.

Significance: The combination of polarized imaging with artificial intelligence (AI) technology has provided a powerful tool for performing an objective and precise diagnosis in medicine.

Aim: An approach is proposed for the detection of hepatitis B (HB) virus using a combined Mueller matrix imaging technique and deep learning method.

Approach: In the proposed approach, Mueller matrix imaging polarimetry is applied to obtain 4 × 4 Mueller matrix images of 138 HBsAg-containing (positive) serum samples and 136 HBsAg-free (negative) serum samples. The kernel estimation density results show that, of the 16 Mueller matrix elements, elements M₂₂ and M₃₃ provide the best discriminatory power between the positive and negative samples.

Results: As a result, M₂₂ and M₃₃ are taken as the inputs to five different deep learning models: Xception, VGG16, VGG19, ResNet 50, and ResNet150. It is shown that the optimal classification accuracy (94.5%) is obtained using the VGG19 model with element M₂₂ as the input.

Conclusions: Overall, the results confirm that the proposed hybrid Mueller matrix imaging and AI framework provides a simple and effective approach for HB virus detection.

Significance: Photoacoustic computed tomography (PACT) is a fast-growing imaging modality. In PACT, the image quality is degraded due to the unknown distribution of the speed of sound (SoS). Emerging initial pressure (IP) and SoS joint-reconstruction methods promise reduced artifacts in PACT. However, previous joint-reconstruction methods have some deficiencies. A more effective method has promising prospects in preclinical applications.

Aim: We propose a multi-segmented feature coupling (MSFC) method for SoS-IP joint reconstruction in PACT.

Approach: In the proposed method, the ultrasound detectors were divided into multiple sub-arrays with each sub-array and its opposite counterpart considered to be a pair. The delay and sum algorithm was then used to reconstruct two images based on a subarray pair and estimated a direction-specific SoS, based on image correlation and the orientation of the subarrays. Once the data generated by all pairs of subarrays were processed, an image that was optimized in terms of minimal feature splitting in all directions was generated. Further, based on the direction-specific SoS, a model-based method was used to directly reconstruct the SoS distribution.

Results: Both phantom and animal experiments demonstrated feasibility and showed promising results compared with conventional methods, with less splitting and blurring and fewer distortions.

Conclusions: The developed MSFC method shows promising results for both IP and SoS reconstruction. The MSFC method will help to optimize the image quality of PACT in clinical applications.

Significance: Light is a good probe for studying the nanoscale-level structural or molecular-specific structural properties of brain cells/tissue due to stress, alcohol, or any other abnormalities. Chronic alcoholism during pregnancy, i.e., fetal alcoholism, being teratogenic, results in fetal alcohol syndrome, and other neurological disorders. Understanding the nano-to-submicron scale spatial structural properties of pup brain cells/tissues using light/photonic probes could provide a plethora of information in understanding the effects of fetal alcoholism.

Aim: Using both light scattering and light localization techniques to probe alterations in nano- to-submicron scale mass density or refractive index fluctuations in brain cells/tissues of mice pups, exposed to fetal alcoholism.

Approach: We use the mesoscopic physics-based dual spectroscopic imaging techniques, partial wave spectroscopy (PWS) and molecular-specific inverse participation ratio (IPR) using confocal imaging, to quantify structural alterations in brain tissues and chromatin/histone in brain cells, respectively, in 60 days postnatal mice pup brain, exposed to fetal alcoholism.

Results: The finer focusing PWS analysis on tissues shows an increase in the degree of structural disorder strength in the pup brain tissues. Furthermore, results of the molecular-specific light localization IPR technique show an increase in the degree of spatial molecular mass density structural disorder in DNA and a decrease in the degree in histone.

Conclusions: In particular, we characterize the spatial pup brain structures from the molecular to tissue levels and address the plausible reasons for such as mass density fluctuations in fetal alcoholism.

Significance: Circulating tumor cells (CTCs) are important biomarkers for cancer management. Isolated CTCs from blood are stained to detect and enumerate CTCs. However, the staining process is laborious and moreover makes CTCs unsuitable for drug testing and molecular characterization.

Aim: The goal is to develop and test deep learning (DL) approaches to detect unstained breast cancer cells in bright-field microscopy images that contain white blood cells (WBCs).

Approach: We tested two convolutional neural network (CNN) approaches. The first approach allows investigation of the prominent features extracted by CNN to discriminate in vitro cancer cells from WBCs. The second approach is based on faster region-based convolutional neural network (Faster R-CNN).

Results: Both approaches detected cancer cells with higher than 95% sensitivity and 99% specificity with the Faster R-CNN being more efficient and suitable for deployment presenting an improvement of 4% in sensitivity. The distinctive feature that CNN uses for discrimination is cell size, however, in the absence of size difference, the CNN was found to be capable of learning other features. The Faster R-CNN was found to be robust with respect to intensity and contrast image transformations.

Conclusions: CNN-based DL approaches could be potentially applied to detect patient-derived CTCs from images of blood samples.

Significance: Imaging needles consist of highly miniaturized focusing optics encased within a hypodermic needle. The needles may be inserted tens of millimeters into tissue and have the potential to visualize diseased cells well beyond the penetration depth of optical techniques applied externally. Multimodal imaging needles acquire multiple types of optical signals to differentiate cell types. However, their use has not previously been demonstrated with live cells.

Aim: We demonstrate the ability of a multimodal imaging needle to differentiate cell types through simultaneous optical coherence tomography (OCT) and fluorescence imaging.

Approach: We characterize the performance of a multimodal imaging needle. This is paired with a fluorescent analog of the therapeutic drug, tamoxifen, which enables cell-specific fluorescent labeling of estrogen receptor-positive (ER+) breast cancer cells. We perform simultaneous OCT and fluorescence in situ imaging on MCF-7 ER+ breast cancer cells and MDA-MB-231 ER− cells. Images are compared against unlabeled control samples and correlated with standard confocal microscopy images.

Results: We establish the feasibility of imaging live cells with these miniaturized imaging probes by showing clear differentiation between cancerous cells.

Conclusions: Imaging needles have the potential to aid in the detection of specific cancer cells within solid tissue.

Significance: Intravascular imaging is key to investigations into atherosclerotic plaque pathobiology and cardiovascular diagnostics overall. The development of multimodal imaging devices compatible with intracoronary applications has the potential to address limitations of currently available single-modality systems.

Aim: We designed and characterized a robust, high performance multimodal imaging system that combines optical coherence tomography (OCT) and multispectral fluorescence lifetime imaging (FLIm) for intraluminal simultaneous assessment of structural and biochemical properties of coronary arteries.

Approach: Several shortcomings of existing FLIm-OCT catheter systems are addressed by adopting key features, namely (1) a custom fiber optic rotary joint based on an air bearing, (2) a broadband catheter using a freeform reflective optics, and (3) integrated solid-state FLIm detectors. Improvements are quantified using a combination of experimental characterization and simulations.

Results: Excellent UV and IR coupling efficiencies and stability (IR: 75.7 % ± 0.4 % , UV: 45.7 % ± 0.35 % ) are achieved; high FLIm optical performance is obtained (UV beam FWHM: 50 μm) contemporaneously with excellent OCT beam quality (IR beam FWHM: 17 μm). High-quality FLIm OCT image of a human coronary artery specimen was acquired.

Conclusion: The ability of this intravascular imaging system to provide comprehensive structural and biochemical properties will be valuable to further our understanding of plaque pathophysiology and improve cardiovascular diagnostics.

Significance: A majority in the photoacoustic (PA) community unconditionally accepts that pulse PA signals show much higher signal-to-noise ratios (SNRs) than continuously excited PA signals. However, we indicate this existing notion would not be valid for very low optical-fluence light-emiting diodes (LEDs)/laser diodes (LDs)-based PA systems.

Aim: We demonstrate in theory and simulation that when the optical fluence of PA-excitation waveforms is much lower than the American National Standards Institute (ANSI) maximum permission exposure (MPE), matched filtered PA signals from chirp waveforms show higher SNRs than those of pulse train waveforms.

Approach: We theoretically derive the PA SNR expression considering the pulse fluence reduction factor based on the ANSI MPE. We investigate and analyze SNR ratios of the pulse train and chirp-waveform matched filtered PA signals with conceptual understanding. We also perform brute-force simulations to extract PA SNRs for the verification of the result.

Results: The brute-force simulations show that the matched filtering with chirp waveforms could achieve better SNRs than pulse train waveforms for very low-fluence PA systems. As the fluence is smaller, the SNR of the matched filtered PA signals is more dominant than that of pulse trains in a wider PA data acquisition time range. In addition, estimated SNR ratios adopting actual parameters of LED/LD-based pulse train PA systems in previous literature support the finding of this paper.

Conclusions: The result can extend the possibility of applying various continuous waveform techniques already studied in the conventional radar technology to PA systems of limited optical power, which would diversify and expedite the research and development of LED/LD-based, compact, and cost-effective PA systems.

Significance: Light-field microscopy has achieved success in various applications of life sciences that require high-speed volumetric imaging. However, existing light-field reconstruction algorithms degrade severely in low-light conditions, and the deconvolution process is time-consuming.

Aim: This study aims to develop a noise robustness phase-space deconvolution method with low computational costs.

Approach: We reformulate the light-field phase-space deconvolution model into the Fourier domain with random-subset ordering and total-variation (TV) regularization. Additionally, we build a time-division-based multicolor light-field microscopy and conduct the three-dimensional (3D) imaging of the heart beating in zebrafish larva at over 95 Hz with a low light dose.

Results: We demonstrate that this approach reduces computational resources, brings a tenfold speedup, and achieves a tenfold improvement for the noise robustness in terms of SSIM over the state-of-the-art approach.

Conclusions: We proposed a phase-space deconvolution algorithm for 3D reconstructions in fluorescence imaging. Compared with the state-of-the-art method, we show significant improvement in both computational effectiveness and noise robustness; we further demonstrated practical application on zebrafish larva with low exposure and low light dose.

Significance: Pulse oximetry is widely used in clinical practice to monitor changes in arterial oxygen saturation (SpO₂). However, decreases in SpO₂ can be delayed relative to the actual clinical event, and near-infrared spectroscopy (NIRS) may detect alterations in oxygenation earlier than pulse oximetry, as shown in previous cerebral oxygenation monitoring studies.

Aim: We aim to compare the response of transcutaneous muscle NIRS measures of the tissue saturation index with pulse oximetry SpO₂ during hypoxia.

Approach: Episodes of acute hypoxia were induced in nine anesthetized Yucatan miniature pigs. A standard pulse oximeter was attached to the ear of the animal, and a transcutaneous NIRS sensor was placed on the hind limb muscle. Hypoxia was induced by detaching the ventilator from the animal and reattaching it once the pulse oximeter reported 70% SpO₂.

Results: Twenty-four episodes of acute hypoxia were analyzed. Upon the start of hypoxia, the transcutaneous NIRS measures changed in 5.3 ± 0.4 s, whereas the pulse oximetry measures changed in 14.9 ± 1.0 s (p < 0.0001).

Conclusions: Transcutaneous muscle NIRS can detect the effects of hypoxia significantly sooner than pulse oximetry in the Yucatan miniature pig. A transcutaneous NIRS sensor may be used as an earlier detector of oxygen saturation changes in the clinical setting than the standard pulse oximeter.

The erratum corrects an error in Fig. 4 of the published article.