Functional and molecular 3D mapping of angiosarcoma tumor using non-invasive laser speckle, hyperspectral, and photoacoustic imaging.

PURPOSE: The gold standard for skin cancer diagnosis is surgical excisional biopsy and histopathological examination. Several non-invasive diagnostic techniques exist, although they have not yet translated into clinical use. This is a proof-of-concept study to assess the possibility of imaging an angiosarcoma in the periocular area. METHODS: We use laser speckle, hyperspectral, and photoacoustic imaging to monitor blood perfusion and oxygen saturation, as well as the molecular composition of the tissue. The information obtained from each imaging modality was combined in order to yield a more comprehensive picture of the function, as well as molecular composition of a rapidly growing cutaneous angiosarcoma in the periocular area. RESULTS: We found an increase in perfusion coupled with a reduction in oxygen saturation in the angiosarcoma. We could also extract the molecular composition of the angiosarcoma at a depth, depicting both the oxygen saturation and highlighting the presence of connective tissue via collagen. CONCLUSIONS: We demonstrate the different physiological parameters that can be obtained with the different techniques and how these can be combined to provide detailed 3D maps of the functional and molecular properties of tumors useful in preoperative assessment.

The effect of traction force on eyelid blood perfusion during closure of defects.

PURPOSE: In oculoplastic surgery the eyelid tissue is frequently stretched in order to repair defects after tumor surgery. However, there is a paucity of research regarding how stretching affects eyelids. The purpose of this study was to gain insight into how traction force affects eyelid stretch as well as tissue perfusion, using a laser-based in vivo monitoring technique. METHOD: Lower-lid pentagonal resections were performed in eight patients and a total of nine eyelids. The medial section of the eyelid was then stretched using a dynamometer up to a force of 2.3 Newtons (N), and eyelid stretching and blood perfusion were continuously measured using laser speckle contrast imaging. RESULTS: Tissue perfusion decreased exponentially when eyelid tissue was stretched, with an initial sharp decline followed by a more gradual reduction. Perfusion approached zero at a force of approximately 2.0 N. The length of the eyelid increased with increasing force up to 1.5 N, after which there was only a very slight increase in length. CONCLUSIONS: Eyelid tissue seems to respond to traction in a non-linear fashion, where the initial force results in the greatest eyelid stretching and reduction in blood perfusion. The results provide information on the effects of a large force for direct closure of large eyelid defects. Considering how quickly perfusion approaches zero, the high success rate of eyelid reconstruction surgery is likely a testament to the extensive vascularization of the periocular region.

Oxygen saturation mapping during reconstructive surgery of human forehead flaps with hyperspectral imaging and spectral unmixing.

BACKGROUND: Optical spectroscopy is commonly used clinically to monitor oxygen saturation in tissue. The most commonly employed technique is pulse oximetry, which provides a point measurement of the arterial oxygen saturation and is commonly used for monitoring systemic hemodynamics, e.g. during anesthesia. Hyperspectral imaging (HSI) is an emerging technology that enables spatially resolved mapping of oxygen saturation in tissue (sO2), but needs to be further developed before implemented in clinical practice. The aim of this study is to demonstrate the applicability of HSI for mapping the sO2 in reconstructive surgery and demonstrate how spectral analysis can be used to obtain clinically relevant sO2 values. METHODS: Spatial scanning HSI was performed on cutaneous forehead flaps, raised as part of a direct brow lift, in eight patients. Pixel-by-pixel spectral analysis, accounting for the absorption from multiple chromophores, was performed and compared to previous analysis techniques to assess sO2. RESULTS: Spectral unmixing using a broad spectral range, and accounting for the absorption of melanin, fat, collagen, and water, provided a more clinically relevant estimate of sO2 than conventional techniques, where typically only spectral features associated with absorption of oxygenated (HbO2) and deoxygenated (HbR) hemoglobin are considered. We demonstrate its clinical applicability by generating sO2 maps of partially excised forehead flaps showed a gradual decrease in sO2 along the length of the flap from 95 % at the flap base to 85 % at the flap tip. After being fully excised, sO2 in the entire flap decreased to 50 % within a few minutes. CONCLUSIONS: The results demonstrate the capability of sO2 mapping in reconstructive surgery in patients using HSI. Spectral unmixing, accounting for multiple chromophores, provides sO2 values that are in accordance with physiological expectations in patients with normal functioning microvascularization. Our results suggest that HSI methods that yield reliable spectra are to be preferred, so that the analysis can produce results that are of clinical relevance.

Hyperspectral and Laser Speckle Contrast Imaging for Monitoring the Effect of Epinephrine in Local Anesthetics in Oculoplastic Surgery.

PURPOSE: Epinephrine is used in local anesthetics to induce vasoconstriction and thus reduce bleeding and prolong the anesthetic effect. Finding the optimal delay between the administration of the anesthetic and skin incision to ensure vasoconstriction and minimize bleeding is important and has recently become the subject of debate. This is the first study to assess blood perfusion and oxygen saturation (sO 2 ) simultaneously in response to a local anesthetic containing epinephrine in human oculoplastic surgery. METHODS: A local anesthetic consisting of lidocaine and epinephrine (20 mg/ml + 12.5 µg/ml) was injected in the eyelids of 9 subjects undergoing blepharoplasty. The perfusion and sO 2 of the eyelids were monitored using laser speckle contrast imaging and hyperspectral imaging, respectively. RESULTS: Laser speckle contrast imaging monitoring showed a decrease in perfusion over time centrally at the site of injection. Half-maximum effect was reached after 34 seconds, and full effect after 115 seconds, determined by exponential fitting. The drop in perfusion decreased gradually further away from the injection site and hypoperfusion was less prominent 4 mm from the injection site, with a spatially dependent half-maximum effect of 231 seconds. Hyperspectral imaging showed only a slight decrease in sO 2 of 11 % at the injection site. CONCLUSIONS: The optimal time delay for skin incision in oculoplastic surgery is approximately 2 minutes after the injection of lidocaine with epinephrine. Longer delay does not lead to a further decrease in perfusion. As sO 2 was only slightly reduced after injection, the results indicate that the use of epinephrine is safe in the periocular region.

Blood Perfusion of Human Upper Eyelid Skin Flaps Is Better in Myocutaneous than in Cutaneous Flaps.

BACKGROUND: The aim of this study was to monitor blood perfusion in human upper eyelid skin flaps and examine how the perfusion is affected by the thickness of the flap. METHODS: Twenty upper eyelids were dissected as part of a blepharoplasty procedure in patients. The medial end of the blepharoplasty flap remained attached to mimic a flap design often used in reconstruction in the periocular area, a myocutaneous flap in which the blood supply follows the fibers of the orbicularis muscle and is thus parallel to the long axis of the flap. The muscle was thereafter dissected from the flap to create a cutaneous flap. Blood perfusion in the 2 types of flaps was compared using laser speckle contrast imaging. RESULTS: Blood perfusion decreased gradually from the base to the tip of all the flaps. Perfusion was significantly higher in the myocutaneous flaps than in the cutaneous flaps (p < 0.0004): 69% in the myocutaneous flaps and 43% in the cutaneous flaps, measured 5 mm from the base. Blood perfusion was preserved to a greater extent distally in the myocutaneous flaps (minimum value seen at 25 mm) than in the cutaneous flaps (minimum seen at 11 mm). CONCLUSIONS: Blood perfusion was better preserved in myocutaneous flaps, including both skin and the orbicularis oculi muscle, than in cutaneous flaps. This may be of clinical interest in patients with poor microcirculation in which a long flap is required for reconstructive surgery.

Giant photoluminescence blinking of perovskite nanocrystals reveals single-trap control of luminescence.

Fluorescence super-resolution microscopy showed correlated fluctuations of photoluminescence intensity and spatial localization of individual perovskite (CH3NH3PbI3) nanocrystals of size ∼200 × 30 × 30 nm(3). The photoluminescence blinking amplitude caused by a single quencher was a hundred thousand times larger than that of a typical dye molecule at the same excitation power density. The quencher is proposed to be a chemical or structural defect that traps free charges leading to nonradiative recombination. These trapping sites can be activated and deactivated by light.

Single Lévy states-disorder induced energy funnels in molecular aggregates.

Using fluorescence super-resolution microscopy we studied simultaneous spectral, spatial localization, and blinking behavior of individual 1D J-aggregates. Excitons migrating 100 nm are funneled to a trap appearing as an additional red-shifted blinking fluorescence band. We propose that the trap is a Frenkel exciton state formed much below the main exciton band edge due to an environmentally induced heavy-tailed Lévy disorder. This points to disorder engineering as a new avenue in controlling light-harvesting in molecular ensembles.

Forehead flap perfusion monitored by laser speckle contrast imaging: Importance of flap length and thickness.

Purpose: Forehead flaps are commonly used in oculoplastic surgery to cover defects after tumor excision. Blood perfusion is vital for flap survival. The aim of this study was to monitor the perfusion in forehead flaps and investigate the impact of flap length and thickness. Methods: Nineteen forehead flaps in patients undergoing direct brow lift were studied. Perfusion was monitored using laser speckle contrast imaging, immediately after raising flaps consisting of epidermis, dermis, and subcutaneous tissue, and after removing the subcutaneous tissue resulting in a thin flap. Results: Perfusion decreased gradually along the length, the mean value being 44% at 5 mm and 26% at 15 mm from the base, in thick flaps. Perfusion was significantly lower in thin flaps, being 13% when measured 15 mm from the flap base (p < 0.0024). Perfusion was better preserved in thick than in thin flaps. Very low perfusion was observed 16.7 mm (16.0-17.3 mm) from the base in thick flaps, and from 10.2 mm (9.8-10.6 mm) from the base in thin flaps (p < 0.0001). Conclusions: Flap thickness is important in maintaining adequate blood perfusion and thus increasing the probability of flap survival. This may be particularly important in long flaps and in patients with impaired microcirculation.

Optimizing clinical O2 saturation mapping using hyperspectral imaging and diffuse reflectance spectroscopy in the context of epinephrine injection.

Clinical determination of oxygen saturation (sO2) in patients is commonly performed via non-invasive optical techniques. However, reliance on a few wavelengths and some form of pre-determined calibration introduces limits to how these methods can be used. One example involves the assessment of sO2 after injection of local anesthetic using epinephrine, where some controversy exists around the time it takes for the epinephrine to have an effect. This is likely caused by a change in the tissue environment not accounted for by standard calibrated instruments and conventional analysis techniques. The present study aims to account for this changing environment by acquiring absorption spectra using hyperspectral imaging (HSI) and diffuse reflectance spectroscopy (DRS) before, during, and after the injection of local anesthesia containing epinephrine in human volunteers. We demonstrate the need to account for multiple absorbing species when applying linear spectral unmixing in order to obtain more clinically relevant sO2 values. In particular, we demonstrate how the inclusion of water absorption greatly affects the rate at which sO2 seemingly drops, which in turn sheds light on the current debate regarding the time required for local anesthesia with epinephrine to have an effect. In general, this work provides important insight into how spectral analysis methods need to be adapted to specific clinical scenarios to more accurately assess sO2.

Facilitating clinically relevant skin tumor diagnostics with spectroscopy-driven machine learning.

In the dawning era of artificial intelligence (AI), health care stands to undergo a significant transformation with the increasing digitalization of patient data. Digital imaging, in particular, will serve as an important platform for AI to aid decision making and diagnostics. A growing number of studies demonstrate the potential of automatic pre-surgical skin tumor delineation, which could have tremendous impact on clinical practice. However, current methods rely on having ground truth images in which tumor borders are already identified, which is not clinically possible. We report a novel approach where hyperspectral images provide spectra from small regions representing healthy tissue and tumor, which are used to generate prediction maps using artificial neural networks (ANNs), after which a segmentation algorithm automatically identifies the tumor borders. This circumvents the need for ground truth images, since an ANN model is trained with data from each individual patient, representing a more clinically relevant approach.

Mapping the architecture of the temporal artery with photoacoustic imaging for diagnosing giant cell arteritis.

Photoacoustic (PA) imaging is rapidly emerging as a promising clinical diagnostic tool. One of the main applications of PA imaging is to image vascular networks in humans. This relies on the signal obtained from oxygenated and deoxygenated hemoglobin, which limits imaging of the vessel wall itself. Giant cell arteritis (GCA) is a treatable, but potentially sight- and life-threatening disease, in which the artery wall is infiltrated by leukocytes. Early intervention can prevent complications making prompt diagnosis of importance. Temporal artery biopsy is the gold standard for diagnosing GCA. We present an approach to imaging the temporal artery using multispectral PA imaging. Employing minimally supervised spectral analysis, we produce histology-like images where the artery wall is clearly discernible from the lumen and further differentiate between PA spectra from biopsies diagnosed as GCA- and GCA+ in 77 patients.

A biophotonic platform for quantitative analysis in the spatial, spectral, polarimetric, and goniometric domains.

Advanced instrumentation and versatile setups are needed for understanding light interaction with biological targets. Such instruments include (1) microscopes and 3D scanners for detailed spatial analysis, (2) spectral instruments for deducing molecular composition, (3) polarimeters for assessing structural properties, and (4) goniometers probing the scattering phase function of, e.g., tissue slabs. While a large selection of commercial biophotonic instruments and laboratory equipment are available, they are often bulky and expensive. Therefore, they remain inaccessible for secondary education, hobbyists, and research groups in low-income countries. This lack of equipment impedes hands-on proficiency with basic biophotonic principles and the ability to solve local problems with applied physics. We have designed, prototyped, and evaluated the low-cost Biophotonics, Imaging, Optical, Spectral, Polarimetric, Angular, and Compact Equipment (BIOSPACE) for high-quality quantitative analysis. BIOSPACE uses multiplexed light-emitting diodes with emission wavelengths from ultraviolet to near-infrared, captured by a synchronized camera. The angles of the light source, the target, and the polarization filters are automated by low-cost mechanics and a microcomputer. This enables multi-dimensional scatter analysis of centimeter-sized biological targets. We present the construction, calibration, and evaluation of BIOSPACE. The diverse functions of BIOSPACE include small animal spectral imaging, measuring the nanometer thickness of a bark-beetle wing, acquiring the scattering phase function of a blood smear and estimating the anisotropic scattering and the extinction coefficients, and contrasting muscle fibers using polarization. We provide blueprints, component list, and software for replication by enthusiasts and educators to simplify the hands-on investigation of fundamental optical properties in biological samples.

Photoacoustic imaging of periorbital skin cancer ex vivo: unique spectral signatures of malignant melanoma, basal, and squamous cell carcinoma.

Radical excision of periorbital skin tumors is difficult without sacrificing excessive healthy tissue. Photoacoustic (PA) imaging is an emerging non-invasive biomedical imagi--ng modality that has potential for intraoperative micrographic control of surgical margins. This is the first study to assess the feasibility of PA imaging for the detection of periocular skin cancer. Eleven patients underwent surgical excision of periocular skin cancer, one of which was a malignant melanoma (MM), eight were basal cell carcinomas (BCCs), and two squamous cell carcinomas (SCCs). Six tumors were located in the eyelid, and five in periocular skin. The excised samples, as well as healthy eyelid samples, were scanned with PA imaging postoperatively, using 59 wavelengths in the range 680-970 nm, to generate 3D multispectral images. Spectral unmixing was performed using endmember spectra for oxygenated and deoxygenated Hb, melanin, and collagen, to iden--tify the chromophore composition of tumors and healthy eyelid tissue. After PA scanning, the tumor samples were examined histopathologically using standard hematoxylin and eosin staining. The PA spectra of healthy eyelid tissue were dominated by melanin in the skin, oxygenated and deoxygenated hemoglobin in the orbicularis oculi muscle, and collagen in the tarsal plate. Multiwavelength 3D scanning provided spectral information on the three tumor types. The spectrum from the MM was primarily reconstructed by the endmember melanin, while the SCCs showed contributions primarily from melanin, but also HbR and collagen. BCCs showed contributions from all four endmembers with a predominance of HbO2 and HbR. PA imaging may be used to distinguish different kinds of periocular skin tumors, paving the way for future intraoperative micrographic control.

Automatic threshold selection algorithm to distinguish a tissue chromophore from the background in photoacoustic imaging.

The adaptive matched filter (AMF) is a method widely used in spectral unmixing to classify different tissue chromophores in photoacoustic images. However, a threshold needs to be applied to the AMF detection image to distinguish the desired tissue chromophores from the background. In this study, we propose an automatic threshold selection (ATS) algorithm capable of differentiating a target from the background, based on the features of the AMF detection image. The mean difference between the estimated thickness, using the ATS algorithm, and the known values was 0.17 SD (0.24) mm for the phantom inclusions and -0.05 SD (0.21) mm for the tissue samples of malignant melanoma. The evaluation shows that the thickness and the width of the phantom inclusions and the tumors can be estimated using AMF in an automatic way after applying the ATS algorithm.

Photoacoustic imaging for the monitoring of local changes in oxygen saturation following an adrenaline injection in human forearm skin.

Clinical monitoring of blood oxygen saturation (sO2) is traditionally performed using optical techniques, such as pulse oximetry and diffuse reflectance spectroscopy (DRS), which lack spatial resolution. Photoacoustic imaging (PAI) is a rapidly developing biomedical imaging technique that is superior to previous techniques in that it combines optical excitation and acoustic detection, providing a map of chromophore distribution in the tissue. Hitherto, PAI has primarily been used in preclinical studies, and only a few studies have been performed in patients. Its ability to measure sO2 with spatial resolution during local vasoconstriction after adrenaline injection has not yet been investigated. Using PAI and spectral unmixing we characterize the heterogeneous change in sO2 after injecting a local anesthetic containing adrenaline into the dermis on the forearm of seven healthy subjects. In comparison to results obtained using DRS, we highlight contrasting results obtained between the two methods arising due to the so-called 'window effect' caused by a reduced blood flow in the superficial vascular plexus. The results demonstrate the importance of spatially resolving sO2 and the ability of PAI to assess the tissue composition in different layers of the skin.

Photoacoustic imaging of the spatial distribution of oxygen saturation in an ischemia-reperfusion model in humans.

Photoacoustic imaging (PAI) is a novel hybrid imaging technique that combines the advantages of optical and ultrasound imaging to produce hyperspectral images of the tissue. The feasibility of measuring oxygen saturation (sO2) with PAI has been demonstrated pre-clinically, but has limited use in humans under conditions of ischemia and reperfusion. As an important step towards making PAI clinically available, we present a study in which PAI was used to estimate the spatial distribution of sO2 in vivo during and after occlusion of the finger of eight healthy volunteers. The results were compared with a commercial oxygen saturation monitor based on diffuse reflectance spectroscopy. We here describe the capability of PAI to provide spatially resolved picture of the evolution of sO2 during ischemia following vascular occlusion of a finger, demonstrating the clinical viability of PAI as a non-invasive diagnostic tool for diseases indicated by impaired microvascularization.

Comparison of photoacoustic imaging and histopathological examination in determining the dimensions of 52 human melanomas and nevi ex vivo.

Surgical excision followed by histopathological examination is the gold standard for the diagnosis and staging of melanoma. Reoperations and unnecessary removal of healthy tissue could be reduced if non-invasive imaging techniques were available for presurgical tumor delineation. However, no technique has gained widespread clinical use to date due to shallow imaging depth or the absence of functional imaging capability. Photoacoustic (PA) imaging is a novel technology that combines the strengths of optical and ultrasound imaging to reveal the molecular composition of tissue at high resolution. Encouraging results have been obtained from previous animal and human studies on melanoma, but there is still a lack of clinical data. This is the largest study of its kind to date, including 52 melanomas and nevi. 3D multiwavelength PA scanning was performed ex vivo, using 59 excitation wavelengths from 680 nm to 970 nm. Spectral unmixing over this broad wavelength range, accounting for the absorption of several tissue chromophores, provided excellent contrast between healthy tissue and tumor. Combining the results of spectral analysis with spatially resolved information provided a map of the tumor borders in greater detail than previously reported. The tumor dimensions determined with PA imaging were strongly correlated with those determined by histopathological examination for both melanomas and nevi.

Compositional and Interfacial Engineering Yield High-Performance and Stable p-i-n Perovskite Solar Cells and Mini-Modules.

Through the optimization of the perovskite precursor composition and interfaces to selective contacts, we achieved a p-i-n-type perovskite solar cell (PSC) with a 22.3% power conversion efficiency (PCE). This is a new performance record for a PSC with an absorber bandgap of 1.63 eV. We demonstrate that the high device performance originates from a synergy between (1) an improved perovskite absorber quality when introducing formamidinium chloride (FACl) as an additive in the "triple cation" Cs0.05FA0.79MA0.16PbBr0.51I2.49 (Cs-MAFA) perovskite precursor ink, (2) an increased open-circuit voltage, VOC, due to reduced recombination losses when using a lithium fluoride (LiF) interfacial buffer layer, and (3) high-quality hole-selective contacts with a self-assembled monolayer (SAM) of [2-(9H-carbazol-9-yl)ethyl]phosphonic acid (2PACz) on ITO electrodes. While all devices exhibit a high performance after fabrication, as determined from current-density voltage, J-V, measurements, substantial differences in device performance become apparent when considering longer-term stability data. A reduced long-term stability of devices with the introduction of a LiF interlayer is compensated for by using FACl as an additive in the metal-halide perovskite thin-film deposition. Optimized devices maintained about 80% of the initial average PCE during maximum power point (MPP) tracking for >700 h. We scaled the optimized device architecture to larger areas and achieved fully laser patterned series-interconnected mini-modules with a PCE of 19.4% for a 2.2 cm2 active area. A robust device architecture and reproducible deposition methods are fundamental for high performance and stable large-area single junction and tandem modules based on PSCs.

Origin of Ionic Inhomogeneity in MAPb(IxBr1-x)3 Perovskite Thin Films Revealed by In-Situ Spectroscopy during Spin Coating and Annealing.

Irradiation-induced phase segregation in mixed methylammonium halide perovskite samples such as methylammonium lead bromide-iodide, MAPb(IxBr1-x)3, is being studied intensively because it limits the efficiency of wide band gap perovskite solar cells. It has been postulated that this phenomenon depends on the intrinsic ionic (in)homogeneity in samples already induced during film formation. A deeper understanding of the MAPb(IxBr1-x)3 formation processes and the influence of the halide ratio, solvents, and the perovskite precursor composition as well as the influence of processing parameters during deposition, e.g., spin coating and annealing parameters, is still lacking. Here, we use a fiber optic-based optical in-situ setup to study the formation processes of the MAPb(IxBr1-x)3 series on a subsecond time scale during spin coating and thermal annealing. In-situ UV-vis measurements during spin coating reveal the influence of different halide ratios, x, in the precursor solution on the preferential crystallization of the phase. Pure bromide samples directly form a perovskite phase, samples with high iodide content form a solvate intermediate phase, and samples with a mixed stoichiometry between 0.1 ≤ x ≤ 0.6 form both. This leads to a heterogeneous formation process via two competing reaction pathways, that leads to a heterogeneous mixture of phases, during spin coating and rationalizes the compositional heterogeneity of mixed bromide-iodide samples even after annealing.

Relating Defect Luminescence and Nonradiative Charge Recombination in MAPbI3 Perovskite Films.

Nonradiative losses in semiconductors are related to defects. At cryogenic temperatures, defect-related photoluminescence (PL) at energies lower than the band-edge PL is observed in methylammonium lead triiodide perovskite. We applied multispectral PL imaging to samples prepared by two different procedures and exhibiting 1 order of magnitude different PL quantum yield (PLQY). The high-PLQY sample showed concentration of the emitting defect sites around 1012-1013 cm-3. No correlation between PLQY and the relative intensity of the defect emission was found when micrometer-sized local regions of the same sample were compared. However, a clear positive correlation between the lower PLQY and higher defect emission was observed when two preparation methods were contrasted. Therefore, although the emissive defects are not connected directly with the nonradiative centers and may be spatially separated at the nano scale, chemical processes during the perovskite synthesis promote/prevent formation of both types of defects at the same time.