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.

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.

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.

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.

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.

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.

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/.

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.

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.

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.

Semi-automated registration and segmentation for gingival tissue volume measurement on 3D OCT images.

The change in gingival tissue volume may be used to indicate changes in gingival inflammation, which may be useful for the clinical assessment of gingival health. Properly quantifying gingival tissue volume requires a robust technique for accurate registration and segmentation of longitudinally captured 3-dimensional (3D) images. In this paper, a semi-automated registration and segmentation method for micrometer resolution measurement of gingival-tissue volume is proposed for 3D optical coherence tomography (OCT) imaging. For quantification, relative changes in gingiva tissue volume are measured based on changes in the gingiva surface height using the tooth surface as a reference. This report conducted repeatability tests on this method drawn from repeated scans in one patient, indicating an error of the point cloud registration method for oral OCT imaging is 63.08 ± 4.52µm (1σ), and the measurement error of the gingival tissue average thickness is -3.40 ± 21.85µm (1σ).

Combined Nd:YAG and Er:YAG lasers for real-time closed-loop tissue-specific laser osteotomy.

A novel real-time and non-destructive method for differentiating soft from hard tissue in laser osteotomy has been introduced and tested in a closed-loop fashion. Two laser beams were combined: a low energy frequency-doubled nanosecond Nd:YAG for detecting the type of tissue, and a high energy microsecond Er:YAG for ablating bone. The working principle is based on adjusting the energy of the Nd:YAG laser until it is low enough to create a microplasma in the hard tissue only (different energies are required to create plasma in different tissue types). Analyzing the light emitted from the generated microplasma enables real-time feedback to a shutter that prevents the Er:YAG laser from ablating the soft tissue.

Interstitial diffuse optical probe with spectral fitting to measure dynamic tumor hypoxia.

Understanding the dynamic nature of tumor hypoxia is vital for cancer therapy. The presence of oxygen within a tumor during radiation therapy increases the likelihood of local control. We used a novel interstitial diffuse optical probe to make real-time measurements of blood volume fraction and hemoglobin oxygen saturation within a tumor at a high temporal resolution. This device was initially characterized and benchmarked using a customized vessel designed to control hemoglobin oxygen saturation and blood volume in a solution of blood with different concentrations of an oxygen scavenger, tetrakis (hydroxymethyl) phosphonium chloride. The optical device was found to consistently monitor the changes in oxygen saturation and these changes correlated to the concentration of the oxygen scavenger added. In near-simultaneous measurements of blood volume and oxygen saturation in tumor-bearing mice, the changes in blood volume fraction and oxygen saturation measured with the interstitial diffuse optical probe were benchmarked against photoacoustic imaging system to track and compare temporal dynamics of oxygen saturation and blood volume in a patient-derived xenograft model of hypopharyngeal carcinoma. Positive correlations between our device and photoacoustic imaging in measuring blood volume and oxygen saturation were observed.

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.

Modeling Tumor Phenotypes In Vitro with Three-Dimensional Bioprinting.

The tumor microenvironment plays a critical role in tumor growth, progression, and therapeutic resistance, but interrogating the role of specific tumor-stromal interactions on tumorigenic phenotypes is challenging within in vivo tissues. Here, we tested whether three-dimensional (3D) bioprinting could improve in vitro models by incorporating multiple cell types into scaffold-free tumor tissues with defined architecture. We generated tumor tissues from distinct subtypes of breast or pancreatic cancer in relevant microenvironments and demonstrate that this technique can model patient-specific tumors by using primary patient tissue. We assess intrinsic, extrinsic, and spatial tumorigenic phenotypes in bioprinted tissues and find that cellular proliferation, extracellular matrix deposition, and cellular migration are altered in response to extrinsic signals or therapies. Together, this work demonstrates that multi-cell-type bioprinted tissues can recapitulate aspects of in vivo neoplastic tissues and provide a manipulable system for the interrogation of multiple tumorigenic endpoints in the context of distinct tumor microenvironments.

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.

Corrigendum: Potential role of the glycolytic oscillator in acute hypoxia in tumors (2015 Phys. Med. Biol. 60 9215).

At the time of publication, our group had performed short tandem repeat (STR) testing on the SCC22B cell line and believed that had been correctly identified. As part of a recent comprehensive process to confirm the identity of cell lines in use in our lab, we repeated STR testing on all cell lines. These results were compared to the ExPASy Cellosaurus database (http://web.expasy.org/cellosaurus/). One cell line used in this manuscript was a near perfect match for T24 (CVCL_0554), a bladder carcinoma cell line commonly found as a cellular contaminant. Although we are unable to test the exact cells used in this manuscript, we believe that the cells labeled as SCC22B are most likely to actually be T24. The authors believe that neither the results nor the conclusions have been significantly changed on the basis of the specific cell line utilized.

Methodological problems in a study of fetal visual perception.

Reid et al.[1] analysed data from 39 third-trimester fetuses, concluding that they showed a preferential head-orienting reaction towards lights projected through the uterine wall in a face-like arrangement, as opposed to an inverted triangle of dots. These results imply not only that assessment of visual-perceptive responses is possible in prenatal subjects, but also that a measurable preference for faces exists before birth. However, we have identified three substantial problems with Reid et al.'s [1] method and analyses, which we outline here.

Optimal wavelength selection for optical spectroscopy of hemoglobin and water within a simulated light-scattering tissue.

An algorithm that selects optimal wavelengths for spectral fitting of diffuse light reflectance spectra using a nonnegative least squares method is presented. Oxyhemoglobin, deoxyhemoglobin, and water are considered representative absorbers, but the approach is not constrained or limited by absorber selection provided native basis spectra are available. The method removes wavelengths iteratively from a scattering-modulated absorption matrix by maximizing the product of its singular values and offers considerable improvements over previously published wavelength selection schemes. Resulting wavelength selections are valid for a broad range of optical properties and yield lower RMS errors than other wavelength combinations. The method is easily modified and broadly applicable to tissue optical spectroscopy. Adaptation of the algorithm to select optimal light-emitting diodes for fitting blood is described.