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

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

Breast Cancer Diagnosis Using Extended-Wavelength-Diffuse Reflectance Spectroscopy (EW-DRS)-Proof of Concept in Ex Vivo Breast Specimens Using Machine Learning.

This study aims to investigate the feasibility of using diffuse reflectance spectroscopy (DRS) to distinguish malignant breast tissue from adjacent healthy tissue, and to evaluate if an extended-wavelength range (450-1550 nm) has an advantage over the standard wavelength range (450-900 nm). Multivariate statistics and machine learning algorithms, either linear discriminant analysis (LDA) or support vector machine (SVM) are used to distinguish the two tissue types in breast specimens (total or partial mastectomy) from 23 female patients with primary breast cancer. EW-DRS has a sensitivity of 94% and specificity of 91% as compared to a sensitivity of 40% and specificity of 71% using the standard wavelength range. The results suggest that DRS can discriminate between malignant and healthy breast tissue, with improved outcomes using an extended wavelength. It is also possible to construct a simple analytical model to improve the diagnostic performance of the DRS technique.

Remote Nanoscopy with Infrared Elastic Hyperspectral Lidar.

Monitoring insects of different species to understand the factors affecting their diversity and decline is a major challenge. Laser remote sensing and spectroscopy offer promising novel solutions to this. Coherent scattering from thin wing membranes also known as wing interference patterns (WIPs) have recently been demonstrated to be species specific. The colors of WIPs arise due to unique fringy spectra, which can be retrieved over long distances. To demonstrate this, a new concept of infrared (950-1650 nm) hyperspectral lidar with 64 spectral bands based on a supercontinuum light source using ray-tracing and 3D printing is developed. A lidar with an unprecedented number of spectral channels, high signal-to-noise ratio, and spatio-temporal resolution enabling detection of free-flying insects and their wingbeats. As proof of principle, coherent scatter from a damselfly wing at 87 m distance without averaging (4 ms recording) is retrieved. The fringed signal properties are used to determine an effective wing membrane thickness of 1412 nm with ±4 nm precision matching laboratory recordings of the same wing. Similar signals from free flying insects (2 ms recording) are later recorded. The accuracy and the method's potential are discussed to discriminate species by capturing coherent features from free-flying insects.

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.

Distinguishing tumor from healthy tissue in human liver ex vivo using machine learning and multivariate analysis of diffuse reflectance spectra.

The aim of this work was to evaluate the capability of diffuse reflectance spectroscopy to distinguish malignant liver tissues from surrounding tissues and to determine whether an extended wavelength range (450-1550 nm) offers any advantages over using the conventional wavelength range. Furthermore, multivariate analysis combined with a machine learning algorithm, either linear discriminant analysis or the more advanced support vector machine, was used to discriminate between and classify freshly excised human liver specimens from 18 patients. Tumors were distinguished from surrounding liver tissues with a sensitivity of 99%, specificity of 100%, classification rate of 100% and a Matthews correlation coefficient of 100% using the extended wavelength range and a combination of principal component analysis and support vector techniques. The results indicate that this technology may be useful in clinical applications for real-time tissue diagnostics of tumor margins where rapid classification is important.

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.

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.

Characterization and modeling of acousto-optic signal strengths in highly scattering media.

Ultrasound optical tomography (UOT) is an imaging technique based on the acousto-optic effect that can perform optical imaging with ultrasound resolution inside turbid media, and is thus interesting for biomedical applications, e.g. for assessing tissue blood oxygenation. In this paper, we present near background free measurements of UOT signal strengths using slow light filter signal detection. We carefully analyze each part of our experimental setup and match measured signal strengths with calculations based on diffusion theory. This agreement between experiment and theory allows us to assert the deep tissue imaging potential of ∼ 5 cm for UOT of real human tissues predicted by previous theoretical studies [Biomed. Opt. Express8, 4523 (2017)] with greater confidence, and indicate that future theoretical analysis of optimized UOT systems can be expected to be reliable.

Extended-wavelength diffuse reflectance spectroscopy with a machine-learning method for in vivo tissue classification.

OBJECTIVES: An extended-wavelength diffuse reflectance spectroscopy (EWDRS) technique was evaluated for its ability to differentiate between and classify different skin and tissue types in an in vivo pig model. MATERIALS AND METHODS: EWDRS recordings (450-1550 nm) were made on skin with different degrees of pigmentation as well as on the pig snout and tongue. The recordings were used to train a support vector machine to identify and classify the different skin and tissue types. RESULTS: The resulting EWDRS curves for each skin and tissue type had a unique profile. The support vector machine was able to classify each skin and tissue type with an overall accuracy of 98.2%. The sensitivity and specificity were between 96.4 and 100.0% for all skin and tissue types. CONCLUSION: EWDRS can be used in vivo to differentiate between different skin and tissue types with good accuracy. Further development of the technique may potentially lead to a novel diagnostic tool for e.g. non-invasive tumor margin delineation.

Clinical Translation of a Novel Photoacoustic Imaging System for Examining the Temporal Artery.

The objective was to provide a clinical setup for photoacoustic imaging (PAI) of the temporal artery in humans and to describe the challenges encountered and methods of overcoming them. The temporal artery was examined in seven patients with suspect giant-cell arteritis (GCA), both in vivo and ex vivo, and the results were compared to that of histology. To adapt PAI to the human studies, the transducer was fixed to an adjustable arm to reduce motion artifacts, and a stepping motor was developed to enable 3-D scanning. Risks associated with the use of lasers, ultrasound, and electrical equipment were evaluated by measuring energy levels, and safety precautions were undertaken to prevent injury to the patients and staff. The PAI spectra obtained clearly delineated the artery wall, both in vivo and ex vivo, although the latter was of high quality due to the lack of artifacts. The results could be compared to that of histology. The involved energy levels were found to be below the limits given in regulatory standards. Eye protectors prevented irradiation of the patient's eyes, and visual function after the procedure was found not to be affected. The patients reported no discomfort during the investigations. PAI provides images of the temporal artery wall that may be used for the future diagnosis of GCA in humans. The technique could be further refined by addressing the specific problems of motion artifacts and interference from blood and other chromophores. This study paves the way for other clinical applications of PAI.

Extended-wavelength diffuse reflectance spectroscopy for a comprehensive view of blood perfusion and tissue response in human forearm skin.

BACKGROUND: The aim of this study was to investigate the possibility of using extended-wavelength diffuse reflectance spectroscopy (EW-DRS) to measure tissue response related to blood perfusion. The study was performed on a model that we have previously found to be useful for studying techniques for perfusion monitoring following the injection of epinephrine in a local anesthetic in the human forearm. METHODS: Nine healthy subjects were included in the study. Spectroscopy was performed with an EW-DRS system using a combination of two spectrometers to resolve light in the visible (350 nm to 1100 nm) and the near-infrared regions (900 nm to 1700 nm). The change in signal upon the injection of lidocaine (20 mg/ml) + epinephrine (12.5 µg/ml) (LIDO +EPI), compared to a control injection with saline (9 mg/ml), was investigated. RESULTS: Injection of lidocaine + epinephrine (12.5 µg/ml) caused a change in the EW-DRS signal in the wavelength intervals 510 to 610 nm, known to change upon deoxygenation of hemoglobin. When examining the full wavelength range (450 to 1550 nm) a decrease in reflectance upon LIDO +EPI injection was observed, suggesting that the broader spectrum provides more detailed information on the tissue response. The time to stable hypoperfusion was found to be 2.6 min. CONCLUSIONS: EW-DRS appears to be a promising technique for monitoring perfusion, and could provide a useful tool in plastic and reconstructive surgery. The broad spectrum provides detailed information on the molecular changes taking place in the tissue. However, the technique must be thoroughly validated before it can be implemented in clinical practice.

Intraoperative liver steatosis characterization using diffuse reflectance spectroscopy.

BACKGROUND: Liver steatosis is associated with poor outcome after liver transplantation and liver resection. There is a need for an accurate and reliable intraoperative tool to identify and quantify steatosis. This study aimed to investigate whether surface diffuse reflectance spectroscopy (DRS) measurements could detect liver steatosis on humans during liver surgery. METHODS: The DRS instrumentation setup consists of a computer, a high-power tungsten halogen light source and two spectrometers, connected through a trifurcated optical fiber to a hand-held probe. Patients scheduled for open resection for liver tumors were considered for inclusion. Multiple DRS measurements were performed on the liver surface after mobilization. RESULTS: In total, 1210 DRS spectra originated from 38 patients, were analyzed. When applying the data to an analytical model the volumetric absorption ratio factor of fat and water specified an explicit distinction between mild to moderate, and moderate to severe steatosis (p < 0.001). There were significant differences between none-to-mild and moderate-to-severe steatosis grade for the following parameters: reduced scattering coefficient (p < 0.001), Mie to total scattering fraction (p < 0.001), Mie slope (p = 0.003), lipid/(lipid + water) (p < 0.001), blood volume (p = 0.044) and bile volume (p < 0.001). CONCLUSION: This study shows that it is possible to evaluate steatosis grades with hepatic surface diffuse reflectance spectroscopy measurements.

Hypoperfusion following the injection of epinephrine in human forearm skin can be measured by RGB analysis but not with laser speckle contrast imaging.

BACKGROUND: The time taken for epinephrine to achieve its optimal effect during local anesthesia has recently become the subject of debate. The time from injection to commencement of surgery is traditionally quoted to be 7 to 10 min, while recent reports claim that it may take 30 min to achieve maximum hypoperfusion, which would prolong the time required for surgical procedures. The discrepancy may be related to difficulties associated with the techniques used to measure blood perfusion. The aim of this study was to test two methods of determining the time to maximum hypoperfusion. METHODS: Laser speckle contrast imaging (LSCI) and red, green, blue (RGB) analysis of images obtained with a commercial digital camera, were used to monitor the effect of infiltration with commonly used local anesthetic preparations: lidocaine (20 mg/ml) + epinephrine (12.5 µg/ml), lidocaine (10 mg/ml) + epinephrine (5 µg/ml), and lidocaine (20 mg/ml) alone, in healthy subjects. RESULTS: LSCI showed a paradoxical increase in signal after the injection of local anesthetics containing epinephrine, probably due to a change in the laser penetration depth resulting from blanching of the skin. However, RGB analysis of digital photographs gave more reliable results, showing skin blanching that corresponded to the expected effect of epinephrine in local anesthetics. The time to maximum effect was found to be 7 (range 5-10) minutes for 12.5 µg/ml epinephrine, and 9 (range 7-13) minutes for 5 µg/ml epinephrine in lidocaine. CONCLUSIONS: RGB analysis of digital images proved to be a valid technique for monitoring the effect of local anesthetics with epinephrine in human skin. The technique requires only a commercial digital camera and constitutes a cheap, simple method. The optimal delay between epinephrine injection and incision, to minimize bleeding, was found to be 7 to 9 min, which is in good agreement with common surgical practice.

Optimal Epinephrine Concentration and Time Delay to Minimize Perfusion in Eyelid Surgery: Measured by Laser-Based Methods and a Novel Form of Extended-Wavelength Diffuse Reflectance Spectroscopy.

OBJECTIVE: This study investigates the hypoperfusion effects of epinephrine in local anesthesia in eyelid surgery. A novel form of extended-wavelength diffuse reflectance spectroscopy was evaluated. METHODS: Blood perfusion in porcine eyelid flaps was measured using laser Doppler velocimetry and laser speckle contrast imaging, whereas the tissue response was measured using diffuse reflectance spectroscopy with a broad spectrum (450-1550 nm). Epinephrine was either injected cumulatively, 0.1 (1:10,000,000), 1.0 (1:1,000,000), 10 (1:100 000), and 100 µg/ml (1:10 000), to determine the dose-response relation, or given as a single dose (10 µg/ml). Control experiments were performed with saline or lidocaine. RESULTS: Increasing concentrations of epinephrine resulted in a gradual decrease in tissue perfusion, measured by laser Doppler velocimetry and laser speckle contrast imaging, approaching a minimum after the injection of 10 µg/ml. Similar tissue response was observed with diffuse reflectance spectroscopy. The time from the injection of epinephrine (10 µg/ml) to the stabilization of hypoperfusion was 75 seconds. After administration of 10 µg/ml epinephrine, about 20% of the blood perfusion remained, supporting the use of epinephrine in eyelid flaps with a narrow pedicle. CONCLUSIONS: 10 µg/ml epinephrine appears to be adequate for vasoconstriction before oculoplastic surgery. Incisions need only be delayed for about 1 minute. Extended-wavelength diffuse reflectance spectroscopy appears to be a promising technique for monitoring the tissue response following changes in blood perfusion in plastic surgery reconstructions. However, more rigorous validation of the technique is required before it can be implemented in clinical practice.

Diffuse Reflectance Spectroscopy for Surface Measurement of Liver Pathology.

BACKGROUND: Liver parenchymal injuries such as steatosis, steatohepatitis, fibrosis, and sinusoidal obstruction syndrome can lead to increased morbidity and liver failure after liver resection. Diffuse reflectance spectroscopy (DRS) is an optical measuring method that is fast, convenient, and established. DRS has previously been used on the liver with an invasive technique consisting of a needle that is inserted into the parenchyma. We developed a DRS system with a hand-held probe that is applied to the liver surface. In this study, we investigated the impact of the liver capsule on DRS measurements and whether liver surface measurements are representative of the whole liver. We also wanted to confirm that we could discriminate between tumor and liver parenchyma by DRS. MATERIALS AND METHODS: The instrumentation setup consisted of a light source, a fiber-optic contact probe, and two spectrometers connected to a computer. Patients scheduled for liver resection due to hepatic malignancy were included, and DRS measurements were performed on the excised liver part with and without the liver capsule and alongside a newly cut surface. To estimate the scattering parameters and tissue chromophore volume fractions, including blood, bile, and fat, the measured diffuse reflectance spectra were applied to an analytical model. RESULTS: In total, 960 DRS spectra from the excised liver tissue of 18 patients were analyzed. All factors analyzed regarding tumor versus liver tissue were significantly different. When measuring through the capsule, the blood volume fraction was found to be 8.4 ± 3.5%, the lipid volume fraction was 9.9 ± 4.7%, and the bile volume fraction was 8.2 ± 4.6%. No differences could be found between surface measurements and cross-sectional measurements. In measurements with/without the liver capsule, the differences in volume fraction were 1.63% (0.75-2.77), -0.54% (-2.97 to 0.32), and -0.15% (-1.06 to 1.24) for blood, lipid, and bile, respectively. CONCLUSION: This study shows that it is possible to manage DRS measurements through the liver capsule and that surface DRS measurements are representative of the whole liver. The results are consistent with data published earlier on the combination of liver chromophores. The results encourage us to proceed with in vivo measurements for further quantification of the liver's composition and assessment of parenchymal damage such as steatosis and fibrosis grade.