Optical Properties of Corals Distort Variable Chlorophyll Fluorescence Measurements.

Pulse-amplitude-modulated (PAM) fluorimetry is widely used in photobiological studies of corals, as it rapidly provides numerous photosynthetic parameters to assess coral ecophysiology. Coral optics studies have revealed the presence of light gradients in corals, which are strongly affected by light scattering in coral tissue and skeleton. We investigated whether coral optics affects variable chlorophyll (Chl) fluorescence measurements and derived photosynthetic parameters by developing planar hydrogel slabs with immobilized microalgae and with bulk optical properties similar to those of different types of corals. Our results show that PAM-based measurements of photosynthetic parameters differed substantially between hydrogels with different degrees of light scattering but identical microalgal density, yielding deviations in apparent maximal electron transport rates by a factor of 2. Furthermore, system settings such as the measuring light intensity affected F 0, Fm , and Fv /Fm in hydrogels with identical light absorption but different degrees of light scattering. Likewise, differences in microalgal density affected variable Chl fluorescence parameters, where higher algal densities led to greater Fv /Fm values and relative electron transport rates. These results have important implications for the use of variable Chl fluorimetry in ecophysiological studies of coral stress and photosynthesis, as well as other optically dense systems such as plant tissue and biofilms.

Hyperspectral imaging in automated digital dermoscopy screening for melanoma.

OBJECTIVES: Early melanoma detection decreases morbidity and mortality. Early detection classically involves dermoscopy to identify suspicious lesions for which biopsy is indicated. Biopsy and histological examination then diagnose benign nevi, atypical nevi, or cancerous growths. With current methods, a considerable number of unnecessary biopsies are performed as only 11% of all biopsied, suspicious lesions are actually melanomas. Thus, there is a need for more advanced noninvasive diagnostics to guide the decision of whether or not to biopsy. Artificial intelligence can generate screening algorithms that transform a set of imaging biomarkers into a risk score that can be used to classify a lesion as a melanoma or a nevus by comparing the score to a classification threshold. Melanoma imaging biomarkers have been shown to be spectrally dependent in Red, Green, Blue (RGB) color channels, and hyperspectral imaging may further enhance diagnostic power. The purpose of this study was to use the same melanoma imaging biomarkers previously described, but over a wider range of wavelengths to determine if, in combination with machine learning algorithms, this could result in enhanced melanoma detection. METHODS: We used the melanoma advanced imaging dermatoscope (mAID) to image pigmented lesions assessed by dermatologists as requiring a biopsy. The mAID is a 21-wavelength imaging device in the 350-950 nm range. We then generated imaging biomarkers from these hyperspectral dermoscopy images, and, with the help of artificial intelligence algorithms, generated a melanoma Q-score for each lesion (0 = nevus, 1 = melanoma). The Q-score was then compared to the histopathologic diagnosis. RESULTS: The overall sensitivity and specificity of hyperspectral dermoscopy in detecting melanoma when evaluated in a set of lesions selected by dermatologists as requiring biopsy was 100% and 36%, respectively. CONCLUSION: With widespread application, and if validated in larger clinical trials, this non-invasive methodology could decrease unnecessary biopsies and potentially increase life-saving early detection events. Lasers Surg. Med. 51:214-222, 2019. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Minimal basilar membrane motion in low-frequency hearing.

Low-frequency hearing is critically important for speech and music perception, but no mechanical measurements have previously been available from inner ears with intact low-frequency parts. These regions of the cochlea may function in ways different from the extensively studied high-frequency regions, where the sensory outer hair cells produce force that greatly increases the sound-evoked vibrations of the basilar membrane. We used laser interferometry in vitro and optical coherence tomography in vivo to study the low-frequency part of the guinea pig cochlea, and found that sound stimulation caused motion of a minimal portion of the basilar membrane. Outside the region of peak movement, an exponential decline in motion amplitude occurred across the basilar membrane. The moving region had different dependence on stimulus frequency than the vibrations measured near the mechanosensitive stereocilia. This behavior differs substantially from the behavior found in the extensively studied high-frequency regions of the cochlea.

Design and calibration of a digital Fourier holographic microscope for particle sizing via goniometry and optical scatter imaging in transmission.

Goniometry and optical scatter imaging have been used for optical determination of particle size based upon optical scattering. Polystyrene microspheres in suspension serve as a standard for system validation purposes. The design and calibration of a digital Fourier holographic microscope (DFHM) are reported. Of crucial importance is the appropriate scaling of scattering angle space in the conjugate Fourier plane. A detailed description of this calibration process is described. Spatial filtering of the acquired digital hologram to use photons scattered within a restricted angular range produces an image. A pair of images, one using photons narrowly scattered within 8 - 15° (LNA), and one using photons broadly scattered within 8 - 39° (HNA), are produced. An image based on the ratio of these two images, OSIR = HNA/LNA, following Boustany et al. (2002), yields a 2D Optical Scatter Image (OSI) whose contrast is based on the angular dependence of photon scattering and is sensitive to the microsphere size, especially in the 0.5-1.0µm range. Goniometric results are also given for polystyrene microspheres in suspension as additional proof of principle for particle sizing via the DFHM.

Methods of Melanoma Detection.

Detection and removal of melanoma, before it has metastasized, dramatically improves prognosis and survival. The purpose of this chapter is to (1) summarize current methods of melanoma detection and (2) review state-of-the-art detection methods and technologies that have the potential to reduce melanoma mortality. Current strategies for the detection of melanoma range from population-based educational campaigns and screening to the use of algorithm-driven imaging technologies and performance of assays that identify markers of transformation. This chapter will begin by describing state-of-the-art methods for educating and increasing awareness of at-risk individuals and for performing comprehensive screening examinations. Standard and advanced photographic methods designed to improve reliability and reproducibility of the clinical examination will also be reviewed. Devices that magnify and/or enhance malignant features of individual melanocytic lesions (and algorithms that are available to interpret the results obtained from these devices) will be compared and contrasted. In vivo confocal microscopy and other cellular-level in vivo technologies will be compared to traditional tissue biopsy, and the role of a noninvasive "optical biopsy" in the clinical setting will be discussed. Finally, cellular and molecular methods that have been applied to the diagnosis of melanoma, such as comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), will be discussed.

The Black Bug Myth: Selective photodestruction of pigmented pathogens.

BACKGROUND AND OBJECTIVE: It is commonly believed that pigmented pathogens are selectively targeted by dental lasers. To test this notion optical diffuse reflection spectroscopy (DRS) was used to obtain absorption spectra for the periodontal pathogens, Porphyromonas gingivalis (Pg) and Prevotella intermedia (Pi). MATERIALS AND METHODS: Spectra from 400 to 1,100 nm wavelengths of Pg colonies cultured with different concentrations of hemin were obtained to test the hypothesis that "visual pigmentation" predicts absorption of near-infrared (IR) dental laser energy. Ablation threshold at 1,064 nm [1] was measured for the pathogenic fungus, Candida albicans (Ca). RESULTS: The hypothesis was demonstrated to be true at 810 nm, it was false at 1,064 nm. Diode laser (810 nm) efficacy and "depth of kill" is dependent on hemin availability from 400 to about 900 nm. Pg and Pi absorption at 1,064 nm (µa = 7.7 ± 2.6 cm(-1) ) is independent of hemin availability but is determined by another unknown chromophore. Ca is non-pigmented but very sensitive to 1,064 nm irradiation. CONCLUSIONS: The amount of visual pigmentation does not necessarily predict sensitivity to dental laser irradiation. Spectra in visible and near-IR wavelengths demonstrate a large difference in absorption between soft tissue and Pg or Pi. This difference represents a host/pathogen differential sensitivity to laser irradiation, the basis for selective photoantisepsis. Lasers Surg. Med. 48:706-714, 2016. © 2016 Wiley Periodicals, Inc.

Filtering of acoustic signals within the hearing organ.

The detection of sound by the mammalian hearing organ involves a complex mechanical interplay among different cell types. The inner hair cells, which are the primary sensory receptors, are stimulated by the structural vibrations of the entire organ of Corti. The outer hair cells are thought to modulate these sound-evoked vibrations to enhance hearing sensitivity and frequency resolution, but it remains unclear whether other structures also contribute to frequency tuning. In the current study, sound-evoked vibrations were measured at the stereociliary side of inner and outer hair cells and their surrounding supporting cells, using optical coherence tomography interferometry in living anesthetized guinea pigs. Our measurements demonstrate the presence of multiple vibration modes as well as significant differences in frequency tuning and response phase among different cell types. In particular, the frequency tuning at the inner hair cells differs from other cell types, causing the locus of maximum inner hair cell activation to be shifted toward the apex of the cochlea compared with the outer hair cells. These observations show that additional processing and filtering of acoustic signals occur within the organ of Corti before inner hair cell excitation, representing a departure from established theories.

Honoring Lihong V. Wang, a pioneer in biomedical optics.

A pioneer in optics based on his development of novel optical imaging techniques and acknowledged by a long list of honors, Lihong V. Wang is a model for the aspiring young student or investigator pursuing a career in the rapidly expanding field of biomedical optics and biophotonics.

Hyperspectral imaging with deep learning for quantification of tissue hemoglobin, melanin, and scattering.

Significance: Hyperspectral cameras capture spectral information at each pixel in an image. Acquired spectra can be analyzed to estimate quantities of absorbing and scattering components, but the use of traditional fitting algorithms over megapixel images can be computationally intensive. Deep learning algorithms can be trained to rapidly analyze spectral data and can potentially process hyperspectral camera data in real time. Aim: A hyperspectral camera was used to capture 1216 × 1936 pixel wide-field reflectance images of in vivo human tissue at 205 wavelength bands from 420 to 830 nm. Approach: The optical properties of oxyhemoglobin, deoxyhemoglobin, melanin, and scattering were used with multi-layer Monte Carlo models to generate simulated diffuse reflectance spectra for 24,000 random combinations of physiologically relevant tissue components. These spectra were then used to train an artificial neural network (ANN) to predict tissue component concentrations from an input reflectance spectrum. Results: The ANN achieved low root mean square errors in a test set of 6000 independent simulated diffuse reflectance spectra while calculating concentration values more than 4000× faster than a conventional iterative least squares approach. Conclusions: In vivo finger occlusion and gingival abrasion studies demonstrate the ability of this approach to rapidly generate high-resolution images of tissue component concentrations from a hyperspectral dataset acquired from human subjects.

IL-23-mediated psoriasis-like epidermal hyperplasia is dependent on IL-17A.

IL-23 and Th17 cells producing IL-17A and IL-22 are found in excess in skin affected by psoriasis. Previous studies showed that IL-22, but not IL-17A, mediates psoriasis-like epidermal hyperplasia following recombinant murine (rm)IL-23 injections into skin. To further investigate the role of IL-17A, ears of mice were injected with rmIL-23. Investigators blinded to treatment conditions and mouse genotypes measured ear swelling, epidermal thickness, and cytokine expression. In wild-type (WT) mice, rmIL-23 induced ear swelling (p < 0.001, all p values versus saline), epidermal hyperplasia by histology (p < 0.001) and confocal microscopy (p < 0.004), and expression of both IL-17A and IL-22. As expected, rmIL-23 injections into IL-22(-/-) mice resulted in relatively little ear swelling (p < 0.09) and epidermal hyperplasia (p < 0.51 by histology and p < 0.75 by confocal microscopy). Notably, rmIL-23 injections into IL-17A(-/-) mice produced little ear swelling (p < 0.001, versus IL-23-injected WT mice) and epidermal hyperplasia (p < 0.001 by histology and p < 0.005 by confocal microscopy), even though IL-22 was readily induced in these mice. Furthermore, systemic delivery of blocking Abs directed against either IL-22 or IL-17A completely inhibited IL-23-induced epidermal hyperplasia in WT mice. These results demonstrate that IL-17A, like IL-22, is a downstream mediator for IL-23-induced changes in murine skin and that both of these Th17 cytokines are necessary to produce IL-23-mediated skin pathology. IL-17A may represent an attractive therapeutic target in individuals with psoriasis by blocking downstream effects of IL-23.

Tutorial on Monte Carlo simulation of photon transport in biological tissues [Invited].

A tutorial introduction to Monte Carlo (MC) simulation of light propagation in biological tissues. MC statistical sampling is introduced, the basic design of a MC program is explained, and examples of application in biomedicine are presented.

Two-term scattering phase function for photon transport to model subdiffuse reflectance in superficial tissues.

For Monte Carlo simulations of light transport in a variety of diffuse scattering applications, a single-scattering two-term phase function with five adjustable parameters is sufficiently flexible to separately control the forward and backward components of scattering. The forward component dominates light penetration into a tissue and the resulting diffuse reflectance. The backward component controls early subdiffuse scatter from superficial tissues. The phase function consists of a linear combination of two phase functions [Reynolds and McCormick, J. Opt. Soc. Am.70, 1206 (1980)10.1364/JOSA.70.001206] that were derived from the generating function for Gegenbauer polynomials. The two-term phase function (TT) accommodates strongly-forward anisotropic scattering with enhanced backscattering and is a generalization of the two-term, three-parameter Henyey-Greenstein phase function. An analytical inverse of the cumulative distribution function for scattering is provided for implementation in Monte Carlo simulations. Explicit TT equations are given for the single-scattering metrics g 1, g 2, γ, and δ. Scattering data from previously published bio-optical data are shown to fit better with the TT than other phase function models. Example Monte Carlo simulations illustrate the use of the TT and its independent control of subdiffuse scatter.

Fiberoptic hemodynamic spectroscopy reveals abnormal cerebrovascular reactivity in a freely moving mouse model of Alzheimer's disease.

Many Alzheimer's disease (AD) patients suffer from altered cerebral blood flow and damaged cerebral vasculature. Cerebrovascular dysfunction could play an important role in this disease. However, the mechanism underlying a vascular contribution in AD is still unclear. Cerebrovascular reactivity (CVR) is a critical mechanism that maintains cerebral blood flow and brain homeostasis. Most current methods to analyze CVR require anesthesia which is known to hamper the investigation of molecular mechanisms underlying CVR. We therefore combined spectroscopy, spectral analysis software, and an implantable device to measure cerebral blood volume fraction (CBVF) and oxygen saturation (SO2) in unanesthetized, freely-moving mice. Then, we analyzed basal CBVF and SO2, and CVR of 5-month-old C57BL/6 mice during hypercapnia as well as during basic behavior such as grooming, walking and running. Moreover, we analyzed the CVR of freely-moving AD mice and their wildtype (WT) littermates during hypercapnia and could find impaired CVR in AD mice compared to WT littermates. Our results suggest that this optomechanical approach to reproducibly getting light into the brain enabled us to successfully measure CVR in unanesthetized freely-moving mice and to find impaired CVR in a mouse model of AD.

Measurement of single cell refractive index, dry mass, volume, and density using a transillumination microscope.

Phase contrast microscopy has become ubiquitous in the field of biology, particularly in qualitative investigations of cellular morphology. However, the use of quantitative phase retrieval methods and their connection to cellular refractive index and dry mass density remain under utilized. This is due in part to the restriction of phase and cellular mass determination to custom built instruments, involved mathematical analysis, and prohibitive sample perturbations. We introduce tomographic bright field imaging, an accessible optical imaging technique enabling the three dimensional measurement of cellular refractive index and dry mass density using a standard transillumination optical microscope. The validity of the technique is demonstrated on polystyrene spheres. The technique is then applied to the measurement of the refractive index, dry mass, volume, and density of red blood cells. This optical technique enables a simple and robust means to perform quantitative investigations of engineered and biological specimens in three dimensions using standard optical microscopes.

History of Monte Carlo modeling of light transport in tissues using mcml.c.

The Monte Carlo simulation called mcml.c was written and shared on-line since 1992. This perspective summarizes the contributions by the people involved in the development of mcml.c, and work by others extending the code.

Spectral imaging of normal, hydrated, and desiccated porcine skin using polarized light.

Significance: Spectroscopic and structural imaging of tissue layers is important for investigating tissue health. However, investigating superficial tissue is difficult using optical imaging, due to the convolved absorption and backscatter of light from deeper layers. Aim: This report investigates the effects of hydration and desiccation of ex vivo porcine skin on the reflectance of polarized light at different wavelengths (light-emitting diodes). Approach: We developed a spectroscopic polarized imaging system to investigate submicron changes in tissue structures. By separating polarized from depolarized backscattered light, submicron structural changes in subsurface and deeper tissue layers can be separated and monitored. Results: The results demonstrate that (1) polarized light reflectance is about 2%, consistent with ∼6 scattering events, on average; (2) there was little wavelength dependence to the reflectance of polarized light; (3) increased hydration leads to a modest increase in total reflectance (from 0.8 to 0.9), whereas desiccation had little effect; however, hydration did not affect polarized reflectance, but desiccation slightly lowered polarized reflectance. Conclusions: Higher scattering from the reticular dermis was likely due to swelling of collagen fiber bundles in the dermal layers, which increased fibril spacing. The epidermal skin surface showed little change due to the stratum corneum resisting desiccation and maintaining hydration.

Graphics-processing-unit-accelerated Monte Carlo simulation of polarized light in complex three-dimensional media.

SIGNIFICANCE: Monte Carlo (MC) methods have been applied for studying interactions between polarized light and biological tissues, but most existing MC codes supporting polarization modeling can only simulate homogeneous or multi-layered domains, resulting in approximations when handling realistic tissue structures. AIM: Over the past decade, the speed of MC simulations has seen dramatic improvement with massively parallel computing techniques. Developing hardware-accelerated MC simulation algorithms that can accurately model polarized light inside three-dimensional (3D) heterogeneous tissues can greatly expand the utility of polarization in biophotonics applications. APPROACH: Here, we report a highly efficient polarized MC algorithm capable of modeling arbitrarily complex media defined over a voxelated domain. Each voxel of the domain can be associated with spherical scatters of various radii and densities. The Stokes vector of each simulated photon packet is updated through photon propagation, creating spatially resolved polarization measurements over the detectors or domain surface. RESULTS: We have implemented this algorithm in our widely disseminated MC simulator, Monte Carlo eXtreme (MCX). It is validated by comparing with a reference central-processing-unit-based simulator in both homogeneous and layered domains, showing excellent agreement and a 931-fold speedup. CONCLUSION: The polarization-enabled MCX offers biophotonics community an efficient tool to explore polarized light in bio-tissues, and is freely available at http://mcx.space/.

Spectral response of optical fiber probe with closely spaced fibers.

BACKGROUND: Optical fiber probe spectroscopy can characterize the blood content, hemoglobin oxygen saturation, water content, and scattering properties of a tissue. A narrow probe using closely spaced fibers can access and characterize a local tissue site, but analysis requires the proper light transport theory. METHODS: Monte Carlo simulations of photon transport specified the response of a two-fiber probe as a function of optical properties in a homogeneous tissue. The simulations used the dimensions of a commercial fiber probe (400-micron-diameter fibers separated by 80-microns of cladding) to calculate the response to a range of 20 absorption and 20 reduced scattering values. The 400 simulations yielded an analysis grid (lookup table) to interpolate the probe response to any given pair of absorption and scattering properties. RESULTS: The probe in contact with tissue is not sensitive to low absorption but sensitive to scattering, as occurs for red to near-infrared spectra. The probe is sensitive to both absorption and scattering for shorter visible spectra (purple-orange). The non-contact probe held above the tissue delivers light to/from a spot on the tissue and fails to collect light that spreads laterally to escape outside the collection spot. Such partial collection can distort the spectra. CONCLUSIONS: Optical fiber spectroscopy using closely spaced fibers requires proper calibration. An analysis subroutine is provided for analysis of a two-fiber probe with the dimensions of a commercial probe (Ocean Insight), but the method can be applied to any probe design. A closely spaced fiber probe can document blood in the shorter visible wavelengths, but has difficulty detecting red and near-infra-red absorption. Hence detection of hydration is difficult. The strength of the closely spaced fiber probe is detecting scattering that depends on tissue structure at the micron to sub-micron scale.

Perspective on diffuse light in tissue: subsampling photon populations.

SIGNIFICANCE: Diffuse light is ubiquitous in biomedical optics and imaging. Understanding the process of migration of an initial photon population entering tissue to a completely randomized, diffusely scattered population provides valuable insight to the interpretation and design of optical measurements. AIM: The goal of this perspective is to present a brief, unifying analytical framework to describe how properties of light transition from an initial state to a distributed state as light diffusion occurs. APPROACH: First, measurement parameters of light are introduced, and Monte Carlo simulations along with a simple analytical expression are used to explore how these individual parameters might exhibit diffusive behavior. Second, techniques to perform optical measurements are considered, highlighting how various measurement parameters can be leveraged to subsample photon populations. RESULTS: Simulation results reinforce the fact that light undergoes a transition from a non-diffuse population to one that is first subdiffuse and then fully diffuse. Myriad experimental methods exist to isolate subpopulations of photons, which can be broadly categorized as source- and/or detector-encoded techniques, as well as methods of tagging the tissue of interest. CONCLUSIONS: Characteristic properties of light progressing to diffusion can be described by some form of Gaussian distribution that grows in space, time, angle, wavelength, polarization, and coherence. In some cases, these features can be approximated by simpler exponential behavior. Experimental methods to subsample features of the photon distribution can be achieved or theoretical methods can be used to better interpret the data with this framework.

Microfluidic photoreactor to treat neonatal jaundice.

While in most cases, jaundice can be effectively treated using phototherapy, severe cases require exchange transfusion, a relatively risky procedure in which the neonate's bilirubin-rich blood is replaced with donor blood. Here, we examine extracorporeal blood treatment in a microfluidic photoreactor as an alternative to exchange transfusion. This new treatment approach relies on the same principle as phototherapy but leverages microfluidics to speed up bilirubin removal. Our results demonstrate that high-intensity light at 470 nm can be used to rapidly reduce bilirubin levels without causing appreciable damage to DNA in blood cells. Light at 470 nm was more effective than light at 505 nm. Studies in Gunn rats show that photoreactor treatment for 4 h significantly reduces bilirubin levels, similar to the bilirubin reduction observed for exchange transfusion and on a similar time scale. Predictions for human neonates demonstrate that this new treatment approach is expected to exceed the performance of exchange transfusion using a low blood flow rate and priming volume, which will facilitate vascular access and improve safety.