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Background: Handheld laser speckle contrast imaging (LSCI) is crucial in clinical settings, but motion artifacts (MA) can compromise perfusion image reliability. Current prevention and suppression methods are often impractical or complex. Machine vision techniques, promising in medical imaging, could improve signal quality, but their use in suppressing MA is still unexplored., Objective: We propose an innovative method based on linear regression for MA correction (MAC) in LSCI and validate it in vivo., Methods: We performed paired handheld and mounted LSCI measurements on 14 subjects with psoriasis using the previously validated handheld perfusion imager (HAPI). By marking lesion boundaries for clinical purposes, the HAPI used a monochromatic camera for both speckle imaging and motion detection, simplifying hardware requirements. Accurate estimation of relative displacements between the test object and LSCI probe allowed us to apply MAC to the perfusion images., Results: Local perfusion values correlated with applied speed were used to calculate and correct MA. The difference between mean perfusion in handheld and mounted modes after MAC significantly decreased (median error 14.2 perfusion units (p.u.) on lesions before correction ( ) and 0.5 p.u. after correction ( p=0.2))., Conclusion: The findings provide evidence for robust handheld LSCI and validate the MA technique in psoriasis case. Of the two causes of MA-onsurface speeds and wavefront tilt-we address the former and correct mean perfusion, assuming constant temporal perfusion at each location., Significance: We describe a practical, non-contact, marker-free technique for reliable handheld perfusion imaging, supporting further clinical translation in plastic surgery and burns.

Significance: In handheld laser speckle contrast imaging (LSCI), motion artifacts (MA) are inevitable. Suppression of MA leads to a valid and objective assessment of tissue perfusion in a wide range of medical applications including dermatology and burns. Our study shines light on the sources of these artifacts, which have not yet been explored. We propose a model based on optical Doppler effect to predict speckle contrast drop as an indication of MA., Aim: We aim to theoretically model MA when an LSCI system measuring on static scattering media is subject to translational displacements. We validate the model using both simulation and experiments. This is the crucial first step toward creating robustness against MA., Approach: Our model calculates optical Doppler shifts in order to predict intensity correlation function and contrast of the time-integrated intensity as functions of applied speed based on illumination and detection wavevectors. To validate the theoretical predictions, computer simulation of the dynamic speckles has been carried out. Then experiments are performed by both high-speed and low-framerate imaging. The employed samples for the experiments are a highly scattering matte surface and a Delrin plate of finite scattering level in which volume scattering occurs., Results: An agreement has been found between theoretical prediction, simulation, and experimental results of both intensity correlation functions and speckle contrast. Coefficients in the proposed model have been linked to the physical parameters according to the experimental setups., Conclusions: The proposed model provides a quantitative description of the influence of the types of illumination and media in the creation of MA. The accurate prediction of MA caused by translation based on Doppler shifts makes our model suitable to study the influence of rotation. Also the model can be extended for the case of dynamic media, such as live tissue., (© 2023 The Authors.)

Clinical measurements on breast cancer patients were performed with a three-dimensional tomographic photoacoustic prototype imager (PAM 2). Patients with a suspicious lesion, visiting the center for breast care of a local hospital, were included in the study. The acquired photoacoustic images were compared to conventional clinical images. Of 30 scanned patients, 19 were diagnosed with one or more malignancies, of which a subset of four patients was selected for detailed analysis. Reconstructed images were processed to enhance image quality and the visibility of blood vessels. Processed photoacoustic images were compared to contrast-enhanced magnetic resonance images where available, which aided in localizing the expected tumoral region. In two cases, spotty high-intensity photoacoustic signals could be seen in the tumoral region, attributable to the tumor. One of these cases also displayed a relatively high image entropy at the tumor site, likely related to the chaotic vascular networks associated with malignancies. For the other two cases, it was not possible to identify features indicative of malignancy, because of limitations in the illumination scheme and difficulties in locating the region of interest in the photoacoustic image., Competing Interests: Co-author T. J. P. M. o. R. is employed by PA Imaging R&D B.V but has no financial interest in the company. Co-author C. K. has a financial interest in PA Imaging Holding B.V., via PAMARA Holding B.V. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors declare that there are no other conflicts of interest., (Copyright: © 2023 Schoustra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Skin microvasculature changes are crucial in psoriasis development and correlate with perfusion. The noninvasive Handheld Perfusion Imager (HAPI) examines microvascular skin perfusion in large body areas using laser speckle contrast imaging (LSCI)., Objectives: To (i) assess whether increased perilesional perfusion and perfusion inhomogeneity are predictors for expansion of psoriasis lesions and (ii) assess feasibility of the HAPI system in a mounted modality., Methods: In this interventional pilot study in adults with unstable plaque psoriasis, HAPI measurements and color photographs were performed for lesions present on one body region at week 0, 2, 4, 6 and 8. The presence of increased perilesional perfusion and perfusion inhomogeneity was determined. Clinical outcome was categorized as increased, stable or decreased lesion surface between visits. Patient feedback was collected on a 10-point scale., Results: In total, 110 lesions with a median follow-up of 6 (IQR 6.0) weeks were assessed in 6 patients with unstable plaque psoriasis. Perfusion data was matched to 281 clinical outcomes after two weeks. A mixed multinomial logistic regression model revealed a predictive value of perilesional increased perfusion (OR 9.90; p < 0.001) and perfusion inhomogeneity (OR 2.39; p = 0.027) on lesion expansion after two weeks compared to lesion stability. HAPI measurements were considered fast, patient-friendly and important by patients., Conclusion: Visualization of increased perilesional perfusion and perfusion inhomogeneity by noninvasive whole field LSCI holds potential for prediction of psoriatic lesion expansion. Furthermore, the HAPI is a feasible and patient-friendly tool., (© 2021 The Authors. Skin Research and Technology published by John Wiley & Sons Ltd.)

We present an acousto-optic imaging system operating in reflection-mode and utilizing a pair of compact, triggerable lasers with 532 and 1064 nm wavelength and nanosecond pulse duration. The system maps the fluence rate distribution of light transmitted through optically scattering samples. The imaging is performed using an acousto-optic probe comprising an ultrasound linear array with attached optical fiber on one side and a camera on the other. The described hardware configuration images samples with access restricted to one side only and ensures mobility of the entire setup. The major challenge of the introduced approach is mitigating the effects of laser parameter instabilities and precise synchronization of ultrasound and laser pulses. We solved this issue by developing an electronic feedback circuit and a microcontroller-based synchronization and control system triggering the ultrasound scanner. Schematics and details regarding control algorithms are introduced. The imaging performance of the system is demonstrated on examples of results obtained for solid, acoustically-homogeneous and optically scattering phantoms with and without light absorbing inclusions present. Adjusting the size and location of the region of interest within the camera sensor matrix and the number of laser pulses illuminating every frame allows for significant improvements in terms of the achievable peak signal to noise ratio. We demonstrate that the developed synchronization algorithm and system play a crucial role in ensuring imaging quality and accuracy., Competing Interests: The authors declare no conflicts of interest., (© 2021 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Fluid flow shear stresses are strong regulators for directing the organization of vascular networks. Knowledge of structural and flow dynamics information within complex vasculature is essential for tuning the vascular organization within engineered tissues, by manipulating flows. However, reported investigations of vascular organization and their associated flow dynamics within complex vasculature over time are limited, due to limitations in the available physiological pre-clinical models, and the optical inaccessibility and aseptic nature of these models. Here, we developed laser speckle contrast imaging (LSCI) and side-stream dark field microscopy (SDF) systems to map the vascular organization, spatio-temporal blood flow fluctuations as well as erythrocytes movements within individual blood vessels of developing chick embryo, cultured within an artificial eggshell system. By combining imaging data and computational simulations, we estimated fluid flow shear stresses within multiscale vasculature of varying complexity. Furthermore, we demonstrated the LSCI compatibility with bioengineered perfusable muscle tissue constructs, fabricated via molding techniques. The presented application of LSCI and SDF on perfusable tissues enables us to study the flow perfusion effects in a non-invasive fashion. The gained knowledge can help to use fluid perfusion in order to tune and control multiscale vascular organization within engineered tissues., (© 2021. The Author(s).)

Significance: Performance of an acousto-optic imaging system is limited by light fluence rate and acoustic pressure field distributions characteristics. In optically scattering media, the former determines the achievable contrast, whereas the latter the imaging resolution. The system parameters can be shaped by changing relative positions of ultrasound (US) transducer array and optodes. However, in the case of many potential clinical applications, optimization possibilities in this regard are limited, as a sample is accessible from one side only and using a water tank for coupling is not feasible., Aim: We investigate the possibilities of improving performance of an acousto-optic imaging system operating in reflection mode geometry with linear US array in direct contact with a sample using plane wave instead of focused US pulses., Approach: Differences in acoustic pressure field distributions for various transducer excitation patterns were determined numerically and experimentally. Acousto-optic images of phantoms with and without optically absorbing inclusions were acquired by measuring laser speckle contrast decrease due to the light modulation by plane wave and focused US pulses with different apodization patterns., Results: The residual acoustic pressure field components occupy relatively large volume and contribute to light modulation. Using nonsteered plane wave US pulses instead of focused ones allows one to mitigate their influence. It also allows one to obtain clear two-dimensional reconstructions of light fluence rate maps by shifting transducer apodization along the lateral direction., Conclusions: Using nonsteered plane wave US pulses allows one to achieve better imaging performance than with focused pulses in the assumed system geometry.

Diagnosis of peripheral artery disease in people with diabetes and a foot ulcer using current non-invasive blood pressure measurements is challenging. Laser speckle contrast imaging (LSCI) is a promising non-invasive technique to measure cutaneous microcirculation. This study investigated the association between microcirculation (measured with both LSCI and non-invasive blood pressure measurement) and healing of diabetic foot ulcers 12 and 26 weeks after measurement. We included sixty-one patients with a diabetic foot ulcer in this prospective, single-center, observational cohort-study. LSCI scans of the foot, ulcer, and ulcer edge were conducted, during baseline and post-occlusion hyperemia. Non-invasive blood pressure measurement included arm, foot, and toe pressures and associated indices. Healing was defined as complete re-epithelialization and scored at 12 and 26 weeks. We found no significant difference between patients with healed or non-healed foot ulcers for both types of measurements ( p = 0.135-0.989). ROC curves demonstrated moderate sensitivity (range of 0.636-0.971) and specificity (range of 0.464-0.889), for LSCI and non-invasive blood pressure measurements. Therefore, no association between diabetic foot ulcer healing and LSCI-measured microcirculation or non-invasive blood pressure measurements was found. The healing tendency of diabetic foot ulcers is difficult to predict based on single measurements using current blood pressure measurements or LSCI.

Enabling handheld perfusion imaging would drastically improve the feasibility of perfusion imaging in clinical practice. Therefore, we examine the performance of handheld laser speckle contrast imaging (LSCI) measurements compared to mounted measurements, demonstrated in psoriatic skin. A pipeline is introduced to process, analyze and compare data of 11 measurement pairs (mounted-handheld LSCI modes) operated on 5 patients and various skin locations. The on-surface speeds (i.e. speed of light beam movements on the surface) are quantified employing mean separation (MS) segmentation and enhanced correlation coefficient maximization (ECC). The average on-surface speeds are found to be 8.5 times greater in handheld mode compared to mounted mode. Frame alignment sharpens temporally averaged perfusion maps, especially in the handheld case. The results show that after proper post-processing, the handheld measurements are in agreement with the corresponding mounted measurements on a visual basis. The absolute movement-induced difference between mounted-handheld pairs after the background correction is [Formula: see text] (mean ± std, [Formula: see text]), with an absolute median difference of [Formula: see text]. Realization of handheld LSCI facilitates measurements on a wide range of skin areas bringing more convenience for both patients and medical staff., (© 2021. The Author(s).)

Oxygen saturation imaging has potential in several preclinical and clinical applications. Dual-wavelength LED array-based photoacoustic oxygen saturation imaging can be an affordable solution in this case. For the translation of this technology, there is a need to improve its accuracy and validate it against ground truth methods. We propose a fluence compensated oxygen saturation imaging method, utilizing structural information from the ultrasound image, and prior knowledge of the optical properties of the tissue with a Monte-Carlo based light propagation model for the dual-wavelength LED array configuration. We then validate the proposed method with oximeter measurements in tissue-mimicking phantoms. Further, we demonstrate in vivo imaging on small animal and a human subject. We conclude that the proposed oxygen saturation imaging can be used to image tissue at a depth of 6-8 mm in both preclinical and clinical applications.

This work describes the design, development and added value of breast-supporting cups to immobilize and position the pendant breast in photoacoustic tomographic imaging. We explain the considerations behind the choice of the material, the shape and sizes of a cup-shaped construct for supporting the breast in water in an imaging tank during full-breast imaging. We provide details of the fabrication, and other processing and testing procedures used. Various experiments were conducted to demonstrate the added value of using these cups. We show that breast movement during a measurement time of four minutes is reduced from maximum 2 mm to 0.1 mm by the use of cups. Further, the presence of the cup, centered in the aperture leading to the imaging tank, ensures that the breast can be reproducibly positioned at the center of the field-of-view of the detection aperture in the tank. Finally, since an accurate delineation of the water-tissue boundary can now be made, the use of the cup enables accurate application of a two-speed of sound model for reconstruction. All in all, we demonstrate that the use of cups to support the breast provides clear enhancement in contrast and resolution of breast images in photoacoustic imaging., Competing Interests: Authors T. J. P. M. o. R., R. P. P. v. M. and L. A. are employed by PA Imaging R&D B.V. but have no financial interest in the company. The authors declare that there are no other conflicts of interest., (© 2020 The Authors.)

Foot ulcers are a severe complication of diabetes mellitus. Assessment of the vascular status of diabetic foot ulcers with Laser Speckle Contrast Imaging (LSCI) is a promising approach for diagnosis and prognosis. However, manual assessment during analysis of LSCI limits clinical applicability. Our aim was to develop and validate a fast and robust tracking algorithm for semi-automatic analysis of LSCI data. The feet of 33 participants with diabetic foot ulcers were recorded with LSCI, including at baseline, during the Post-Occlusive Reactive Hyperemia (PORH) test, and during the Buerger's test. Different regions of interest (ROIs) were used to measure microcirculation in different areas of the foot. A tracking algorithm was developed in MATLAB to reposition the ROIs in the LSCI scans. Manual- and algorithm-tracking of all recordings were compared by calculating the Intraclass Correlation Coefficient (ICC). The algorithm was faster in comparison with the manual approach (90 s vs. 15 min). Agreement between manual- and algorithm-tracking was good to excellent during baseline (ICC = 0.896-0.984; p < 0.001), the PORH test (ICC = 0.790-0.960; p < 0.001), and the Buerger's test (ICC = 0.851-0.978; p < 0.001), resulting in a tracking algorithm that delivers assessment of LSCI in diabetic foot ulcers with results comparable to a labor-intensive manual approach, but with a 10-fold workload reduction.

One of the remaining challenges of bringing photoacoustic imaging to clinics is the occurrence of reflection artifacts. Previously, we proposed a method using multi-wavelength excitation to identify and remove the RAs. However, this method requires at least 3 wavelengths. Here we improve the method further by reducing the required number of wavelengths to 2. We experimentally demonstrate this new method and compare it with the previous one. Results show that this new method holds great feasibility for identifying reflection artifacts in addition to preserving all advantages of the previous method., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Significance: Practical implementation of acousto-optic imaging (AOI) encounters difficulties that prevent it from rapid adoption in clinical use. In many practical medical applications, the region of interest may be accessed only from one side, and using a water tank for coupling is not feasible. The solution might be to use reflection-mode imaging with an electronically scanned ultrasound (US) focus. Such an approach, however, entails considerable challenges., Aim: The possibilities of detecting and localizing light-absorbing inclusions inside turbid media by combining reflection-mode AOI conducted using a one-dimensional US array with electronic scanning of the US focus are investigated experimentally and signal processing algorithms that could be used for this purpose are introduced., Approach: We determine the speckle contrast decrease due to the acousto-optic effect as a function of the US focal point coordinates. Different signal postprocessing techniques are investigated., Results: A significant decrease in the determined speckle contrast difference values is observed due to the presence of light-absorbing inclusions. However, local minima occur in the plots only under specific conditions. Subtracting individual distributions and determining symmetry deviations allow for localizing the inclusions., Conclusions: Detection and localization of optically distinct regions are possible using the introduced approach. Signal postprocessing is required in a general case.

Small animals are widely used as disease models in medical research. Noninvasive imaging modalities with functional capability play an important role in studying the disease state and treatment progress. Photoacoustics, being a noninvasive and functional modality, has the potential for small-animal imaging. However, the conventional photoacoustic tomographic systems use pulsed lasers, making it expensive, bulky, and require long acquisition time. In this work, we propose the use of photoacoustic and ultrasound tomographic imaging with LEDs as the light source and acoustic detection using a linear transducer array. We have demonstrated full-view tomographic imaging of a euthanized mouse and a potential application in liver fibrosis research.

Functional performance of handheld laser speckle contrast imaging (LSCI) is compromised by movement artefacts. Here we quantify the movements of a handheld LSCI system employing electromagnetic (EM) tracking and measure the applied translational, tilt and on-surface laser beam speeds. By observing speckle contrast on static objects, the magnitudes of translation and tilt of wavefronts are explored for various scattering levels of the objects. We conclude that for tissue mimicking static phantoms, on-surface speeds play a dominant role to wavefront tilt speed in creation of movement artefacts. The ratio depends on the optical properties of the phantom. Furthermore, with the same applied speed, the drop in the speckle contrast increases with decreasing reduced scattering coefficient, and hence the related movement artefact increases., Competing Interests: The authors declare that there are not any financial interests related to this article and no potential conflicts of interest to disclose., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Significance: Quantifying human milk composition is important for daily nutritional management in neonatal intensive cares worldwide. Photonic solutions based on visible light can potentially aid in this analysis, as energy content of human milk depends largely on fat content, and the optical scattering properties of human milk predominantly depend on the size and concentration of fat globules. However, it is expected that human milk scattering changes upon homogenization, routinely done before analysis, which may affect fat globule size., Aim: The first aim of this study was to investigate how the most common homogenization methods (gently inverting by hand, vortexing, and sonication) affect the optical properties of human milk. The second aim was to estimate the scattering contribution of casein micelles, the second most dominant scatterers in human milk., Approach: We combined diffuse reflectance spectroscopy with spectroscopic optical coherence tomography to measure the scattering coefficient μs, reduced scattering coefficient μs', and anisotropy g between 450 and 600 nm., Results: Sonication induced the strongest changes in μs, μs', and g compared to the gently inverted samples (203%, 202%, and 7%, respectively, at 550 nm), but also vortexing changed μs' with 20%. Although casein micelles only showed a modest contribution to μs and g at 550 nm (7% and 1%, respectively), their contribution to μs' was 29%., Conclusions: The scattering properties of human milk strongly depend on the homogenization method that is employed, and gentle inversion should be the preferred method. The contribution of casein micelles was relatively small for μs and g but considerably larger for μs'.

Pulsed lasers in photoacoustic tomography systems are expensive, which limit their use to a few clinics and small animal labs. We present a method to realize tomographic ultrasound and photoacoustic imaging using a commercial LED-based photoacoustic and ultrasound system. We present two illumination configurations using LED array units and an optimal number of angular views for tomographic reconstruction. The proposed method can be a cost-effective solution for applications demanding tomographic imaging and can be easily integrated into conventional linear array-based ultrasound systems. We present a potential application for finger joint imaging in vivo , which can be used for point-of-care rheumatoid arthritis diagnosis and monitoring., Competing Interests: M. K. A. S is employed by CYBERDYNE Inc. The authors have no financial interests or conflict of interest to disclose., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Research on photoacoustic imaging (PAI) using a handheld integrated photoacoustic probe has been a recent focus of clinical translation of this imaging technique. One of the remaining challenges is the occurrence of out-of-plane artifacts (OPAs) in such a probe. Previously, we proposed a method to identify and remove OPAs by axially displacing the transducer array. Here we show that besides the benefit of removing OPAs from the imaging plane, the proposed method can provide a three-dimensional (3D) view of the OPAs. In this work, we present a 3D reconstruction method using axial transducer array displacement. By axially displacing the transducer array, out-of-plane absorbers can be three-dimensionally visualized at an elevation distance of up to the acquired imaging depth. Additionally, OPAs in the in-plane image are significantly reduced. We experimentally demonstrate the method with phantom and in vivo experiments using an integrated PAI probe. We also compare the method with elevational transducer array displacement and take into account the sensitivity of the transducer array in the 3D reconstruction., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

The non-invasive quantification of total haemoglobin concentrations [tHb] is highly desired for the assessment of haematologic disorders in vulnerable patient groups, but invasive blood sampling is still the gold standard in current clinical practice. This work demonstrates the potential of visible-light spectroscopic optical coherence tomography (sOCT) for quantifying the [tHb] in human whole blood. To accurately quantify the [tHb] from the substantial optical attenuation by blood in the visible wavelength range, we used a combination of zero-delay acquisition and focus tracking that ensures optimal system sensitivity at any depth inside the sample. Subsequently, we developed an analysis model to adequately correct for the high scattering contribution by red blood cells to the sOCT signal. We validate our method and compare it to conventional sOCT (without focus tracking and zero-delay acquisition) through ex-vivo measurements on flowing human whole blood, with [tHb] values in the clinical range of 7-23 g/dL. For our method with optimized sensitivity, the measured and expected values correlate well (Pearson correlation coefficient = 0.89, p < 0.01), with a precision of 3.8 g/dL. This is a considerable improvement compared to conventional sOCT (Pearson correlation coefficient = 0.59, p = 0.16; precision of 9.1 g/dL).

We present the Twente Photoacoustic Mammoscope 2, a photoacoustic breast imaging system employing a tomographic configuration. It images one breast pendant inside an imaging tank filled with water while a woman lies prone on a bed. A dual-head laser (755 and 1064 nm) illuminates the breast with one beam directed at the nipple and nine beams directed at the sides. Ultrasound signals are detected using 12 arc-shaped arrays, each curving along the pendant breast. Each array comprises 32 piezocomposite elements each with a center frequency of 1 MHz. The imaging tank and the ultrasound arrays rotate around the breast in steps to obtain additional multiple projections. Three-dimensional images are reconstructed using a filtered backprojection algorithm. The system is described in detail, and measurements on a test object are presented. As part of a preliminary study to assess the system's in vivo performance, the breasts of two healthy volunteers were imaged. These images show the breast contour, the nipple, and the vascular anatomy within the breast. In the nipple of one case, multiple high-intensity "hot spots" are observed, which we suspect are associated with the lactiferous ducts terminating in the nipple.

When a biological tissue is illuminated with coherent light, an interference pattern will be formed at the detector, the so-called speckle pattern. Laser speckle contrast imaging (LSCI) is a technique based on the dynamic change in this backscattered light as a result of interaction with red blood cells. It can be used to visualize perfusion in various tissues and, even though this technique has been extensively described in the literature, the actual clinical implementation lags behind. We provide an overview of LSCI as a tool to image tissue perfusion. We present a brief introduction to the theory, review clinical studies from various medical fields, and discuss current limitations impeding clinical acceptance.

With human milk being the most important source of infant nutrition, the protection and support of breastfeeding are essential from a global health perspective. Nevertheless, relatively few objective methods are available to investigate human milk composition and lactation physiology when a mother experiences breastfeeding problems. We argue that optics and photonics offer promising opportunities for this purpose. Any research activity within this new application field starts with a thorough understanding on how light interacts with human milk. Therefore, the aim of this study was to investigate the full set of optical properties for human milk and the biological variability therein. Using a novel approach that combines spatially resolved diffuse reflectance spectroscopy (SR-DRS) and spectroscopic optical coherence tomography (sOCT) between 450 and 650 nm, we quantified the absorption coefficient µ a , scattering coefficient µ s , reduced scattering coefficient µ s ' , anisotropy g and backscattering coefficient µ b,NA of mature human milk from 14 participants released at different stages during a breastfeed (foremilk, bulk milk and hindmilk). Significant correlations were found between µ a , µ s , µ s ' and µ b,NA and the biochemically determined fat concentration per sample ( R s = 0.38, R s = 0.77, R s = 0.80, R s = 0.44 respectively). We explained the observed variations in the optical properties of human milk using Mie theory and the biological variability in both the concentration and size distribution of milk fat globules. In conclusion, we have provided a full set of optical properties for human milk, which can hopefully serve as a starting point for future biophotonic studies on human milk and the milk containing lactating breast., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

Objective: A major challenge for treating diabetic foot ulcers is estimating the severity of ischemia, as the currently used non-invasive diagnostic techniques provide relatively poor prognostic values. Laser speckle contrast imaging (LSCI) is a promising non-invasive technique to assess microcirculation. Our aim was to investigate the stability and reproducibility of LSCI for the assessment of microcirculation in the diabetic foot, the relation of LSCI results to currently used non-invasive blood pressure measurements, and the ability of LSCI to discriminate between the degrees of ischemia., Approach: Thirty-three participants with diabetic foot ulcers were included in this prospective, single centre, observational cohort study that was conducted in the Netherlands. They were classified as non-ischemic, ischemic or critical-ischemic based on criteria formulated in the international guidelines. Two clinicians performed LSCI scans of the foot, consisting of baseline measurements, followed by two stress tests (post-occlusion peak and elevation test). With three measurement conditions and five regions of interest of the foot per patient, a total of 15 measurements were available for analyses., Main Results: The intra-observer agreement of LSCI was high (interclass correlation coefficient (ICC)  =  0.711-0.950; p   <  0.001) for all 15 measurements. The inter-observer agreement was high (ICC  =  0.728-0.861; p   ⩽  0.001) for 10 measurements and moderate (ICC  =  0.476-0.570; p   ⩽  0.005) for the remaining five measurements. The inter-assessor agreement was high and significant (ICC  =  0.857-0.996; p   ⩽  0.001) for all measurements. Correlation between LSCI and non-invasive blood pressure measurements was low (ICC  =  -0.272-0.582). During both stress tests, microcirculation was significantly lower in critical-ischemic feet compared to non-ischemic feet (67.5 perfusion units (PU) versus 96.3 PU and 41.0 PU versus 63.9 PU; p   <  0.05)., Significance: LSCI is a stable and reproducible technique for assessment of microcirculation in people with diabetic foot ulcers and shows significant differences between non-ischemic, ischemic and critical-ischemic patient populations.

Objective: Knowledge on the hemodynamics of the human lactating breast contributes to our understanding of lactation physiology, as well as the development and management of breastfeeding problems. The objective of this pilot study was to investigate whether laser Doppler perfusion monitoring (LDPM) can be employed to measure physiological changes in mammary cutaneous perfusion during milk extraction., Approach: We evaluated mammary cutaneous perfusion with LDPM in nine lactating women during milk extraction in both the ipsilateral ('milk extracting') and contralateral ('passive') breast. Fourier domain filtering of the LDPM signal was applied to correct for the influence of the periodic tissue movement caused by the breast pump., Main Results: Cutaneous perfusion increased temporary during 23.7  ±  18.9 s by 18%-74% for all women who sensed their milk ejection reflex (n  =  6) in both the ipsilateral and contralateral breast. For those women who did not sense a milk ejection reflex (n  =  3), the changes in cutaneous perfusion were less outspoken (maximally 26%)., Significance: This pilot study provides new insights into mammary hemodynamics and demonstrates that LDPM is a promising method for the further investigation of physiological changes in mammary cutaneous perfusion during milk ejection. Objective feedback on the occurrence and progression of milk ejection can support lactation research in general, maternal breastfeeding confidence, and may be an early indicator for the development of breastfeeding problems.

The occurrence of artifacts is a major challenge in photoacoustic imaging. The artifacts negatively affect the quality and reliability of the images. An approach using multi-wavelength excitation has previously been reported for in-plane artifact identification. Yet, out-of-plane artifacts cannot be tackled with this method. Here we propose a new method using ultrasound transducer array displacement. By displacing the ultrasound transducer array axially, we can de-correlate out-of-plane artifacts with in-plane image features and thus remove them. Combining this new method with the previous one allows us to remove potentially completely both in-plane and out-of-plane artifacts in photoacoustic imaging. We experimentally demonstrate this with experiments in phantoms as well as in vivo., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

Photoacoustic imaging has been a focus of research for clinical applications owing to its ability for deep visualization with optical absorption contrast. However, there are various technical challenges remaining for this technique to find its place in clinics. One of the challenges is the occurrence of reflection artifacts. The reflection artifacts may lead to image misinterpretation. Here we propose a new method using multiple wavelengths for identifying and removing the reflection artifacts. By imaging the sample with multiple wavelengths, the spectral response of the features in the photoacoustic image is obtained. We assume that the spectral response of the reflection artifact is better correlated with the proper image feature of its corresponding absorber than with other features in the image. Based on this, the reflection artifacts can be identified and removed. Here, we experimentally demonstrated the potential of this method for real-time identification and correction of reflection artifacts in photoacoustic images in phantoms as well as in vivo using a handheld photoacoustic imaging probe., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

The deep imaging capability and optical absorption contrast offered by photoacoustic imaging promote the use of this technology in clinical applications. By exploiting the optical absorption properties of endogenous chromophores such as hemoglobin and lipid, molecular information at a depth of a few centimeters can be unveiled. This information shows promise to reveal lesions indicating early stage of various human diseases, such as cancer and atherosclerosis. In addition, the use of exogenous contrast agents can further extend the capability of photoacoustic imaging in clinical diagnosis and treatment. In this review, the current state of the art and applications of photoacoustic molecular probes will be critically reviewed, as well as some spearheading translational efforts that have taken place over the past 5 years. Some of the most critical barriers to clinical translation of this novel technology will be discussed, and some thoughts will be given on future endeavors and pathways., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

Spatially confined measurements of bilirubin in tissue can be of great value for noninvasive bilirubin estimations during neonatal jaundice, as well as our understanding of the physiology behind bilirubin extravasation. This work shows the potential of spectroscopic visible-light optical coherence tomography (sOCT) for this purpose. At the bilirubin absorption peak around 460 nm, sOCT suffers from a strong signal decay with depth, which we overcome by optimizing our system sensitivity through a combination of zero-delay acquisition and focus tracking. In a phantom study, we demonstrate the quantification of bilirubin concentrations between 0 and 650 µM with only a 10% difference to the expected value, thereby covering the entire clinical pathophysiological range., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

Photoacoustic tomography is a hybrid imaging technique that combines high optical tissue contrast with high ultrasound resolution. Direct reconstruction methods such as filtered back-projection, time reversal and least squares suffer from curved line artefacts and blurring, especially in the case of limited angles or strong noise. In recent years, there has been great interest in regularised iterative methods. These methods employ prior knowledge of the image to provide higher quality reconstructions. However, easy comparisons between regularisers and their properties are limited, since many tomography implementations heavily rely on the specific regulariser chosen. To overcome this bottleneck, we present a modular reconstruction framework for photoacoustic tomography, which enables easy comparisons between regularisers with different properties, e.g. nonlinear, higher-order or directional. We solve the underlying minimisation problem with an efficient first-order primal-dual algorithm. Convergence rates are optimised by choosing an operator-dependent preconditioning strategy. A variety of reconstruction methods are tested on challenging 2D synthetic and experimental data sets. They outperform direct reconstruction approaches for strong noise levels and limited angle measurements, offering immediate benefits in terms of acquisition time and quality. This work provides a basic platform for the investigation of future advanced regularisation methods in photoacoustic tomography.

Photoacoustic flowmetry (PAF) based on time-domain cross correlation of photoacoustic signals is a promising technique for deep tissue measurement of blood flow velocity. Signal processing has previously been developed for single element transducers. Here, the processing methods for acoustic resolution PAF using a clinical ultrasound transducer array are developed and validated using a 64-element transducer array with a -6 dB detection band of 11 to 17 MHz. Measurements were performed on a flow phantom consisting of a tube (580  μm inner diameter) perfused with human blood flowing at physiological speeds ranging from 3 to 25  mm  /  s. The processing pipeline comprised: image reconstruction, filtering, displacement detection, and masking. High-pass filtering and background subtraction were found to be key preprocessing steps to enable accurate flow velocity estimates, which were calculated using a cross-correlation based method. In addition, the regions of interest in the calculated velocity maps were defined using a masking approach based on the amplitude of the cross-correlation functions. These developments enabled blood flow measurements using a transducer array, bringing PAF one step closer to clinical applicability., ((2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Background: The most severe diabetic foot ulcers are those related with critical ischemia, which is primarily diagnosed with non-invasive diagnostics. However, these diagnostics have several disadvantages. For example, they only provide global indications of the (macro)level of ischemia. A potential solution can be found in novel optical imaging techniques for local assessment of the microcirculation in diabetic foot ulcers. This review provides an overview of these imaging techniques (Laser Doppler Perfusion Imaging, Laser Speckle Contrast Imaging, Photoacoustic Imaging and Hyperspectral Imaging) and their applicability for the diagnostic assessment of microcirculation in diabetic foot ulcers., Method: For each technique, the following parts are described: a) their technical background; b) general clinical applications; and, c) its application for microcirculation assessment in diabetic foot ulcers. Parts a-b are based on a narrative review of the literature, part c on a systematic review that was performed in the database Scopus, covering the period from January 1, 2000 to November 31, 2017., Results: Each of these techniques has specific advantages and disadvantages for imaging microcirculation. Potential clinical use depends on measurement aims, and clinical relevance. However, none of the techniques has a strongly established clinical relevance yet: we found a limited number of publications describing clinical outcomes. Future research is needed to determine which technique is the most clinically relevant for the assessment of microcirculation in diabetic foot ulcers., Conclusion: Although promising, the currently available novel optical techniques need to be further improved technically and prospective trials are necessary to evaluate their clinical value., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

We report the use of ultrasound modulated optical tomography (UOT) with heterodyne parallel detection to locally sense and image blood flow deep inside a highly scattering medium. We demonstrate that the UOT signal is sensitive to the speed of the blood flow in the ultrasound focus and present an analytical model that relates UOT signals to the optical properties (i. e. scattering coefficient, anisotropy, absorption, and flow speed) of the blood and the background medium. We found an excellent agreement between the experimental data and the analytical model. By varying the integration time of the camera in our setup, we were able to spatially resolve blood flow in a scattering medium with a lateral resolution of 1.5 mm., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We evaluate a portable ultrasound and photoacoustic imaging (PAI) system for the feasibility of a point-of-care assessment of clinically evident synovitis. Inflamed and non-inflamed proximal interphalangeal joints of 10 patients were examined and compared with joints from 7 healthy volunteers. PAI scans, ultrasound power Doppler (US-PD), and clinical examination were performed. We quantified the amount of photoacoustic (PA) signal using a region of interest (ROI) drawn over the hypertrophic joint space. PAI response was increased 4 to 10 fold when comparing inflamed with contralateral non-inflamed joints and with joints from healthy volunteers (p < 0.001 for both). US-PD and PAI were strongly correlated (Spearman's ρ = 0.64, with 95% CI: 0.42, 0.79). Hence, PAI using a compact handheld probe is capable of detecting clinically evident synovitis. This motivates further investigation into the predictive value of PAI, including multispectral PAI, with other established modalities such as US-PD or MRI.

Quantitative photoacoustic imaging in biomedicine relies on accurate measurements of relevant material properties of target absorbers. Here, we present a method for simultaneous measurements of the absorption coefficient and Grüneisen parameter of small volume of liquid scattering and absorbing media using a coupled-integrating sphere system which we refer to as quantitative photoacoustic integrating sphere (QPAIS) platform. The derived equations do not require absolute magnitudes of optical energy and pressure values, only calibration of the setup using aqueous ink dilutions is necessary. As a demonstration, measurements with blood samples from various human donors are done at room and body temperatures using an incubator. Measured absorption coefficient values are consistent with known oxygen saturation dependence of blood absorption at 750 nm, whereas measured Grüneisen parameter values indicate variability among five different donors. An increasing Grüneisen parameter value with both hematocrit and temperature is observed. These observations are consistent with those reported in literature.

Reflection artifacts caused by acoustic inhomogeneities constitute a major problem in epi-mode biomedical photoacoustic imaging. Photoacoustic transients from the skin and superficial optical absorbers traverse into the tissue and reflect off echogenic structures to generate reflection artifacts. These artifacts cause difficulties in the interpretation of images and reduce contrast and imaging depth. We recently developed a method called PAFUSion (photoacoustic-guided focused ultrasound) to circumvent the problem of reflection artifacts in photoacoustic imaging. We already demonstrated that the photoacoustic signals can be backpropagated using synthetic aperture pulse-echo data for identifying and reducing reflection artifacts in vivo . In this work, we propose an alternative variant of PAFUSion in which synthetic backpropagation of photoacoustic signals is based on multi-angled plane-wave ultrasound measurements. We implemented plane-wave and synthetic aperture PAFUSion in a handheld ultrasound/photoacoustic imaging system and demonstrate reduction of reflection artifacts in phantoms and in vivo measurements on a human finger using both approaches. Our results suggest that, while both approaches are equivalent in terms of artifact reduction efficiency, plane-wave PAFUSion requires less pulse echo acquisitions when the skin absorption is the main cause of reflection artifacts.

An important problem in minimally invasive photoacoustic (PA) imaging of brachytherapy seeds is reflection artifacts caused by the high signal from the optical fiber/needle tip reflecting off the seed. The presence of these artifacts confounds interpretation of images. In this letter, we demonstrate a recently developed concept called photoacoustic-guided focused ultrasound (PAFUSion) for the first time in the context of interstitial illumination PA imaging to identify and remove reflection artifacts. In this method, ultrasound (US) from the transducer is focused on the region of the optical fiber/needle tip identified in a first step using PA imaging. The image developed from the US diverging from the focus zone at the tip region visualizes only the reflections from seeds and other acoustic inhomogeneities, allowing identification of the reflection artifacts of the first step. These artifacts can then be removed from the PA image. Using PAFUSion, we demonstrate reduction of reflection artifacts and thereby improved interstitial PA visualization of brachytherapy seeds in phantom and

Liver fibrosis is a major cause for increasing mortality worldwide. Preclinical research using animal models is required for the discovery of new anti-fibrotic therapies, but currently relies on endpoint liver histology. In this study, we investigated a cost-effective and portable photoacoustic/ultrasound (PA/US) imaging system as a potential non-invasive alternative. Fibrosis was induced in mice using CCl 4 followed by liver imaging and histological analysis. Imaging showed significantly increased PA features with higher frequency signals in fibrotic livers versus healthy livers. This corresponds to more heterogeneous liver structure resulting from collagen deposition and angiogenesis. Importantly, PA response and its frequency were highly correlated with histological parameters. These results demonstrate the preclinical feasibility of the PA imaging approach and applicability of dual PA/US system.

Objectives: Photoacoustic mammography is potentially an ideal technique, however, the amount of patient data is limited. To further our understanding of the in vivo performance of the method and to guide further research and development, we imaged 33 breast malignancies using the research system - the Twente Photoacoustic Mammoscope (PAM)., Methods: Thirty-one patients participated in this retrospective, observational study. The study and informed consent procedure were approved by the local ethics committee. PAM uses 1,064 nm light for excitation with a planar, 588-element, 1-MHz ultrasound array for detection. Photoacoustic lesion visibility and appearance were compared with conventional imaging (x-ray mammography and ultrasonography) findings, histopathology and patient demographics., Results: Of 33 malignancies 32 were visualized with high contrast and good co-localization with conventional imaging. The contrast of the detected malignancies was independent of radiographic breast density, and size estimation was reasonably good with an average 28 % deviation from histology. However, the presence of contrast areas outside the malignant region is suggestive for low specificity of the current system. Statistical analyses did not reveal any further relationship between PAM results and patient demographics nor lesion characteristics., Conclusions: The results confirm the high potential of photoacoustic mammography in future breast care., Key Points: • Photoacoustic breast imaging visualizes malignancies with high imaging contrast. • Photoacoustic lesion contrast is independent of the mammographically estimated breast density. • No clear relationship exists between photoacoustic characteristics and lesion type, grade, etc. • Photoacoustic specificity to breast cancer from some cases is not yet optimal.

Reflection artifacts caused by acoustic inhomogeneities are a critical problem in epi-mode biomedical photoacoustic imaging. High light fluence beneath the probe results in photoacoustic transients, which propagate into the tissue and reflect back from echogenic structures. These reflection artifacts cause problems in image interpretation and significantly impact the contrast and imaging depth. We recently proposed a method called PAFUSion (Photoacoustic-guided focused ultrasound) to identify such reflection artifacts in photoacoustic imaging. In its initial version, PAFUSion mimics the inward-travelling wavefield from small blood vessel-like PA sources by applying ultrasound pulses focused towards these sources, and thus provides a way to identify the resulting reflection artifacts. In this work, we demonstrate reduction of reflection artifacts in phantoms and in vivo measurements on human volunteers. In view of the spatially distributed PA sources that are found in clinical applications, we implemented an improved version of PAFUSion where photoacoustic signals are backpropagated to imitate the inward travelling wavefield and thus the reflection artifacts. The backpropagation is performed in a synthetic way based on the pulse-echo acquisitions after transmission on each single element of the transducer array. The results provide a direct confirmation that reflection artifacts are prominent in clinical epi-photoacoustic imaging, and that PAFUSion can strongly reduce these artifacts to improve deep-tissue photoacoustic imaging.

The noninvasive and longitudinal imaging of cells or cell aggregates in large optically scattering scaffolds is still a largely unresolved problem in tissue engineering. In this work, we investigated the potential of near-infrared (NIR) photoacoustic (PA) tomography imaging to address this issue. We used clinically relevant sizes of highly light scattering polyethersulfone multibore(®) hollow fiber scaffolds seeded with cells. Since cells have little optical absorption at NIR wavelengths, we studied labeling of cells with absorbers. Four NIR labels were examined for their suitability based on absorption characteristics, resistance to bleaching, and influence on cell viability. On the basis of these criteria, carbon nanoparticles proved most suitable in a variety of cells. For PA imaging, we used a research setup, based on computed tomography geometry. As proof of principle, using this imager we monitored the distribution and clustering of labeled rat insulinoma beta cell aggregates in the scaffolds. This was performed for the duration of 1 week in a nondestructive manner. The results were validated using fluorescence imaging, histology, and light microscopy imaging. Based on our findings, we conclude that PA tomography is a powerful tool for the nondestructive imaging of cells in optically scattering tissue-engineered scaffolds.

In photoacoustic spectroscopy (PAS), wavelength dependent optical attenuation of biological tissue presents a challenge to measure the absolute oxygen saturation of hemoglobin (sO2). Here, we employ the combination of photoacoustics and acousto-optics (AO) at two optical wavelengths to achieve quantification, where AO serves as a sensor for the relative local fluence. We demonstrate that our method enables compensation of spatial as well as wavelength dependent fluence variations in PAS without a priori knowledge about the optical properties of the medium. The fluence compensated photoacoustic images at two excitation wavelengths are used to estimate the absolute oxygen saturation of blood in a spatially and spectroscopically heterogeneous phantom.

A method for measuring the absorption coefficient μ a of absorbing and scattering liquid samples is presented. The sample is injected into a small transparent tube mounted through an integrating sphere. Two models for determining the absorption coefficient using the relative optical output signal are described and validated using aqueous ink absorbers of 0.5 vol.% (0.3  mm -1 <μ a <1.55  mm -1 ) and 1.0 vol.% (1.0  mm -1 <μ a <4.0  mm -1 ) concentrations with 1 vol.% (μs'≈1.4  mm -1 ) and 10 vol.% (μs'≈14  mm -1 ) Intralipid dilutions. The low concentrations give μ a and μ s values, which are comparable with those of biological tissues. One model assumes a uniform light distribution within the sample, which is valid for low absorption. Another model considers light attenuation that obeys Lambert-Beer's law, which may be used for relatively high absorption. Measurements with low and high scattering samples are done for the wavelength range of 400-900 nm. Measured spectra of purely absorbing samples are within 15% agreement with measurements using standard transmission spectrophotometry. For 0.5 vol.% ink absorbers and at wavelengths below 700 nm, measured μ a values are higher for samples with low scattering and lower for those with high scattering. At wavelengths above 700 nm, measured μ a values do not vary significantly with amount of scattering. For 1.0 vol.% ink absorbers, measured spectra do not change with low scattering. These results indicate that the method can be used for measuring absorption spectra of scattering liquid samples with optical properties similar to biological absorbers, particularly at wavelengths above 700 nm, which is difficult to accomplish with standard transmission spectrophotometry.

Flow imaging is an important technique in a range of disease areas, but estimating low flow speeds, especially near the walls of blood vessels, remains challenging. Pulsed photoacoustic flow imaging can be an alternative since there is little signal contamination from background tissue with photoacoustic imaging. We propose flow imaging using a clinical photoacoustic system that is both handheld and portable. The system integrates a linear array with 7.5 MHz central frequency in combination with a high-repetition-rate diode laser to allow high-speed photoacoustic imaging--ideal for this application. This work shows the flow imaging performance of the system in vitro using microparticles. Both two-dimensional (2-D) flow images and quantitative flow velocities from 12 to 75  mm/s were obtained. In a transparent bulk medium, flow estimation showed standard errors of ∼7% the estimated speed; in the presence of tissue-realistic optical scattering, the error increased to 40% due to limited signal-to-noise ratio. In the future, photoacoustic flow imaging can potentially be performed in vivo using fluorophore-filled vesicles or with an improved setup on whole blood.

Recently we presented novel methods for acousto-optic (AO) imaging of biological tissues, taking (1) the mean square difference of speckle patterns (subtraction method) or (2) the contrast of the summation of speckle patterns (summation method) acquired from nanosecond pulses of coherent light, fired at different ultrasound phases. In this study we relate the two methods both analytically and experimentally. We experimentally show that these two methods are nearly identical provided that the maximum achievable speckle contrast is determined correctly. We show with simulations that after correction the outcome is independent of experimental detection parameters. This makes the AO methods in this study reliable, allowing quantifying speckle observations in terms of the ultrasonically tagged fractions of light. The use of tandem nanosecond pulses in one burst of ultrasound overcomes the challenge of tissue dynamics.

Photoacoustic (PA) imaging of interphalangeal peripheral joints is of interest in the context of using the synovial membrane as a surrogate marker of rheumatoid arthritis. Previous work has shown that ultrasound (US) produced by absorption of light at the epidermis reflects on the bone surfaces within the finger. When the reflected signals are backprojected in the region of interest, artifacts are produced, confounding interpretation of the images. In this work, we present an approach where the PA signals known to originate from the epidermis are treated as virtual US transmitters, and a separate reconstruction is performed as in US reflection imaging. This allows us to identify the bone surfaces. Furthermore, the identification of the joint space is important as this provides a landmark to localize a region-of-interest in seeking the inflamed synovial membrane. The ability to delineate bone surfaces allows us to identify not only the artifacts but also the interphalangeal joint space without recourse to new US hardware or a new measurement. We test the approach on phantoms and on a healthy human finger., (© The Author(s) 2015.)

Influence of acoustic inhomogeneities and resulting reflection artifacts is an important problem in reflection-mode photoacoustic imaging. Absorption of light by skin and superficial optical absorbers will generate photoacoustic transients, which traverse into the tissue and get reflected from structures having different acoustic impedance. These reflected photoacoustic signals, when reconstructed, may appear in the region of interest, which causes difficulties in image interpretation. We propose a novel method to identify and potentially eliminate reflection artifacts in photoacoustic images using photoacoustic-guided focused ultrasound [PAFUSion]. Our method uses focused ultrasound pulses to mimic the wave field produced by photoacoustic sources and thus provides a way to identify reflection artifacts in clinical combined photoacoustic and pulse-echo ultrasound. Simulation and phantom results are presented to demonstrate the validity and impact of this method. Results show that PAFUSion can identify reflections in photoacoustic images and thus envisages potential for improving photoacoustic imaging of acoustically inhomogeneous tissue.