Multi-frequency spatial frequency domain imaging: a depth-resolved optical scattering model to isolate scattering contrast in thin layers of skin.

Significance: Current methods for wound healing assessment rely on visual inspection, which gives qualitative information. Optical methods allow for quantitative non-invasive measurements of optical properties relevant to wound healing. Aim: Spatial frequency domain imaging (SFDI) measures the absorption and reduced scattering coefficients of tissue. Typically, SFDI assumes homogeneous tissue; however, layered structures are present in skin. We evaluate a multi-frequency approach to process SFDI data that estimates depth-specific scattering over differing penetration depths. Approach: Multi-layer phantoms were manufactured to mimic wound healing scattering contrast in depth. An SFDI device imaged these phantoms and data were processed according to our multi-frequency approach. The depth sensitive data were then compared with a two-layer scattering model based on light fluence. Results: The measured scattering from the phantoms changed with spatial frequency as our two-layer model predicted. The performance of two δ-P1 models solutions for SFDI was consistently better than the standard diffusion approximation. Conclusions: We presented an approach to process SFDI data that returns depth-resolved scattering contrast. This method allows for the implementation of layered optical models that more accurately represent physiologic parameters in thin tissue structures as in wound healing.

MicroRNA-155 mediates multiple gene regulations pertinent to the role of human adipose-derived mesenchymal stem cells in skin regeneration.

Introduction: The role of Adipose-derived mesenchymal stem cells (AD-MSCs) in skin wound healing remains to be fully characterized. This study aims to evaluate the regenerative potential of autologous AD-MSCs in a non-healing porcine wound model, in addition to elucidate key miRNA-mediated epigenetic regulations that underlie the regenerative potential of AD-MSCs in wounds. Methods: The regenerative potential of autologous AD-MSCs was evaluated in porcine model using histopathology and spatial frequency domain imaging. Then, the correlations between miRNAs and proteins of AD-MSCs were evaluated using an integration analysis in primary human AD-MSCs in comparison to primary human keratinocytes. Transfection study of AD-MSCs was conducted to validate the bioinformatics data. Results: Autologous porcine AD-MSCs improved wound epithelialization and skin properties in comparison to control wounds. We identified 26 proteins upregulated in human AD-MSCs, including growth and angiogenic factors, chemokines and inflammatory cytokines. Pathway enrichment analysis highlighted cell signalling-associated pathways and immunomodulatory pathways. miRNA-target modelling revealed regulations related to genes encoding for 16 upregulated proteins. miR-155-5p was predicted to regulate Fibroblast growth factor 2 and 7, C-C motif chemokine ligand 2 and Vascular cell adhesion molecule 1. Transfecting human AD-MSCs cell line with anti-miR-155 showed transient gene silencing of the four proteins at 24 h post-transfection. Discussion: This study proposes a positive miR-155-mediated gene regulation of key factors involved in wound healing. The study represents a promising approach for miRNA-based and cell-free regenerative treatment for difficult-to-heal wounds. The therapeutic potential of miR-155 and its identified targets should be further explored in-vivo.

Evaluation of the robustness of cerebral oximetry to variations in skin pigmentation using a tissue-simulating phantom.

Clinical studies have demonstrated that epidermal pigmentation level can affect cerebral oximetry measurements. To evaluate the robustness of these devices, we have developed a phantom-based test method that includes an epidermis-simulating layer with several melanin concentrations and a 3D-printed cerebrovascular module. Measurements were performed with neonatal, pediatric and adult sensors from two commercial oximeters, where neonatal probes had shorter source-detector separation distances. Referenced blood oxygenation levels ranged from 30 to 90%. Cerebral oximeter outputs exhibited a consistent decrease in saturation level with simulated melanin content; this effect was greatest at low saturation levels, producing a change of up to 15%. Dependence on pigmentation was strongest in a neonatal sensor, possibly due to its high reflectivity. Overall, our findings indicate that a modular channel-array phantom approach can provide a practical tool for assessing the impact of skin pigmentation on cerebral oximeter performance and that modifications to algorithms and/or instrumentation may be needed to mitigate pigmentation bias.

Water and hemoglobin modulated gelatin-based phantoms to spectrally mimic inflamed tissue in the validation of biomedical techniques and the modeling of microdialysis data.

SIGNIFICANCE: Tissue simulating phantoms are an important part of validating biomedical optical techniques. Tissue pathology in inflammation and oedema involves changes in both water and hemoglobin fractions. AIM: We present a method to create solid gelatin-based phantoms mimicking inflammation and oedema with adjustable water and hemoglobin fractions. APPROACH: One store-bought gelatin and one research grade gelatin were evaluated. Different water fractions were obtained by varying the water-to-gelatin ratio. Ferrous stabilized human hemoglobin or whole human blood was added as absorbers, and the stability and characteristics of each were compared. Intralipid® was used as the scatterer. All phantoms were characterized using spatial frequency domain spectroscopy. RESULTS: The estimated water fraction varied linearly with expected values (R2 = 0.96 for the store-bought gelatin and R2 = 0.99 for the research grade gelatin). Phantoms including ferrous stabilized hemoglobin stayed stable up to one day but had methemoglobin present at day 0. The phantoms with whole blood remained stable up to 3 days using the store-bought gelatin. CONCLUSIONS: A range of physiological relevant water fractions was obtained for both gelatin types, with the stability of the phantoms including hemoglobin differing between the gelatin type and hemoglobin preparation. These low-cost phantoms can incorporate other water-based chromophores and be fabricated as thin sheets to form multilayered structures.

Spectral characterization of liquid hemoglobin phantoms with varying oxygenation states.

SIGNIFICANCE: For optical methods to accurately assess hemoglobin oxygen saturation in vivo, an independently verifiable tissue-like standard is required for validation. For this purpose, we propose three hemoglobin preparations and evaluate methods to characterize them. AIM: To spectrally characterize three different hemoglobin preparations using multiple spectroscopic methods and to compare their absorption spectra to commonly used reference spectra. APPROACH: Absorption spectra of three hemoglobin preparations in solution were characterized using spectroscopic collimated transmission: whole blood, lysed blood, and ferrous-stabilized hemoglobin. Tissue-mimicking phantoms composed of Intralipid, and the hemoglobin solutions were characterized using spatial frequency-domain spectroscopy (SFDS) and enhanced perfusion and oxygen saturation (EPOS) techniques while using yeast to deplete oxygen. RESULTS: All hemoglobin preparations exhibited similar absorption spectra when accounting for methemoglobin and scattering in their oxyhemoglobin and deoxyhemoglobin forms, respectively. However, systematic differences were observed in the fitting depending on the reference spectra used. For the tissue-mimicking phantoms, SFDS measurements at the surface of the phantom were affected by oxygen diffusion at the interface with air, associated with higher values than for the EPOS system. CONCLUSIONS: We show the validity of different blood phantoms and what considerations need to be addressed in each case to utilize them equivalently.

Characterization of nanosensitive multifractality in submicron scale tissue morphology and its alteration in tumor progression.

SIGNIFICANCE: Assessment of disease using optical coherence tomography is an actively investigated problem, owing to many unresolved challenges in early disease detection, diagnosis, and treatment response monitoring. The early manifestation of disease or precancer is typically associated with subtle alterations in the tissue dielectric and ultrastructural morphology. In addition, biological tissue is known to have ultrastructural multifractality. AIM: Detection and characterization of nanosensitive structural morphology and multifractality in the tissue submicron structure. Quantification of nanosensitive multifractality and its alteration in progression of tumor. APPROACH: We have developed a label free nanosensitive multifractal detrended fluctuation analysis(nsMFDFA) technique in combination with multifractal analysis and nanosensitive optical coherence tomography (nsOCT). The proposed method deployed for extraction and quantification of nanosensitive multifractal parameters in mammary fat pad (MFP). RESULTS: Initially, the nsOCT approach is numerically validated on synthetic submicron axial structures. The nsOCT technique was applied to pathologically characterized MFP of murine breast tissue to extract depth-resolved nanosensitive submicron structures. Subsequently, two-dimensional MFDFA were deployed on submicron structural en face images to extract nanosensitive tissue multifractality. We found that nanosensitive multifractality increases in transition from healthy to tumor. CONCLUSIONS: This method for extraction of nanosensitive tissue multifractality promises to provide a noninvasive diagnostic tool for early disease detection and monitoring treatment response. The novel ability to delineate the dominant submicron scale nanosensitive multifractal properties may also prove useful for characterizing a wide variety of complex scattering media of non-biological origin.

Characterization of nanosensitive multifractality in submicron scale tissue morphology and its alteration in tumor progression (Erratum).

An error in the 2nd author's name is corrected.

Handheld multispectral imager for quantitative skin assessment in low-resource settings.

SIGNIFICANCE: Spatial frequency domain imaging (SFDI) is a quantitative imaging method to measure absorption and scattering of tissue, from which several chromophore concentrations (e.g., oxy-/deoxy-/meth-hemoglobin, melanin, and carotenoids) can be calculated. Employing a method to extract additional spectral bands from RGB components (that we named cross-channels), we designed a handheld SFDI device to account for these pigments, using low-cost, consumer-grade components for its implementation and characterization. AIM: With only three broad spectral bands (red, green, blue, or RGB), consumer-grade devices are often too limited. We present a methodology to increase the number of spectral bands in SFDI devices that use RGB components without hardware modification. APPROACH: We developed a compact low-cost RGB spectral imager using a color CMOS camera and LED-based mini projector. The components' spectral properties were characterized and additional cross-channel bands were calculated. An alternative characterization procedure was also developed that makes use of low-cost equipment, and its results were compared. The device performance was evaluated by measurements on tissue-simulating optical phantoms and in-vivo tissue. The measurements were compared with another quantitative spectroscopy method: spatial frequency domain spectroscopy (SFDS). RESULTS: Out of six possible cross-channel bands, two were evaluated to be suitable for our application and were fully characterized (520 ± 20 nm; 556 ± 18 nm). The other four cross-channels presented a too low signal-to-noise ratio for this implementation. In estimating the optical properties of optical phantoms, the SFDI data have a strong linear correlation with the SFDS data (R2 = 0.987, RMSE = 0.006 for µa, R2 = 0.994, RMSE = 0.078 for µs'). CONCLUSIONS: We extracted two additional spectral bands from a commercial RGB system at no cost. There was good agreement between our device and the research-grade SFDS system. The alternative characterization procedure we have presented allowed us to measure the spectral features of the system with an accuracy comparable to standard laboratory equipment.

Impact of hemoglobin breakdown products in the spectral analysis of burn wounds using spatial frequency domain spectroscopy.

Burn wounds and wound healing invoke several biological processes that may complicate the interpretation of spectral imaging data. Through analysis of spatial frequency domain spectroscopy data (450 to 1000 nm) obtained from longitudinal investigations using a graded porcine burn wound healing model, we have identified features in the absorption spectrum that appear to suggest the presence of hemoglobin breakdown products, e.g., methemoglobin. Our results show that the calculated concentrations of methemoglobin directly correlate with burn severity, 24 h after the injury. In addition, tissue parameters such as oxygenation (StO2) and water fraction may be underestimated by 20% and 78%, respectively, if methemoglobin is not included in the spectral analysis.

Method using in vivo quantitative spectroscopy to guide design and optimization of low-cost, compact clinical imaging devices: emulation and evaluation of multispectral imaging systems.

With recent proliferation in compact and/or low-cost clinical multispectral imaging approaches and commercially available components, questions remain whether they adequately capture the requisite spectral content of their applications. We present a method to emulate the spectral range and resolution of a variety of multispectral imagers, based on in-vivo data acquired from spatial frequency domain spectroscopy (SFDS). This approach simulates spectral responses over 400 to 1100 nm. Comparing emulated data with full SFDS spectra of in-vivo tissue affords the opportunity to evaluate whether the sparse spectral content of these imagers can (1) account for all sources of optical contrast present (completeness) and (2) robustly separate and quantify sources of optical contrast (crosstalk). We validate the approach over a range of tissue-simulating phantoms, comparing the SFDS-based emulated spectra against measurements from an independently characterized multispectral imager. Emulated results match the imager across all phantoms (<3 % absorption, <1 % reduced scattering). In-vivo test cases (burn wounds and photoaging) illustrate how SFDS can be used to evaluate different multispectral imagers. This approach provides an in-vivo measurement method to evaluate the performance of multispectral imagers specific to their targeted clinical applications and can assist in the design and optimization of new spectral imaging devices.

Evaluation of a pointwise microcirculation assessment method using liquid and multilayered tissue simulating phantoms.

A fiber-optic probe-based instrument, designed for assessment of parameters related to microcirculation, red blood cell tissue fraction (fRBC), oxygen saturation (SO2), and speed resolved perfusion, has been evaluated using state-of-the-art tissue phantoms. The probe integrates diffuse reflectance spectroscopy (DRS) at two source-detector separations and laser Doppler flowmetry, using an inverse Monte Carlo method for identifying the parameters of a multilayered tissue model. Here, we characterize the accuracy of the DRS aspect of the instrument using (1) liquid blood phantoms containing yeast and (2) epidermis-dermis mimicking solid-layered phantoms fabricated from polydimethylsiloxane, titanium oxide, hemoglobin, and coffee. The root-mean-square (RMS) deviations for fRBC for the two liquid phantoms were 11% and 5.3%, respectively, and 11% for the solid phantoms with highest hemoglobin signatures. The RMS deviation for SO2 was 5.2% and 2.9%, respectively, for the liquid phantoms, and 2.9% for the solid phantoms. RMS deviation for the reduced scattering coefficient (µs'), for the solid phantoms was 15% (475 to 850 nm). For the liquid phantoms, the RMS deviation in average vessel diameter (D) was 1 µm. In conclusion, the skin microcirculation parameters fRBC and SO2, as well as, µs' and D are estimated with reasonable accuracy.

Portable (handheld) clinical device for quantitative spectroscopy of skin, utilizing spatial frequency domain reflectance techniques.

Spatial Frequency Domain Spectroscopy (SFDS) is a technique for quantifying in-vivo tissue optical properties. SFDS employs structured light patterns that are projected onto tissues using a spatial light modulator, such as a digital micromirror device. In combination with appropriate models of light propagation, this technique can be used to quantify tissue optical properties (absorption, µa, and scattering, µs', coefficients) and chromophore concentrations. Here we present a handheld implementation of an SFDS device that employs line (one dimensional) imaging. This instrument can measure 1088 spatial locations that span a 3 cm line as opposed to our original benchtop SFDS system that only collects a single 1 mm diameter spot. This imager, however, retains the spectral resolution (∼1 nm) and range (450-1000 nm) of our original benchtop SFDS device. In the context of homogeneous turbid media, we demonstrate that this new system matches the spectral response of our original system to within 1% across a typical range of spatial frequencies (0-0.35 mm-1). With the new form factor, the device has tremendously improved mobility and portability, allowing for greater ease of use in a clinical setting. A smaller size also enables access to different tissue locations, which increases the flexibility of the device. The design of this portable system not only enables SFDS to be used in clinical settings but also enables visualization of properties of layered tissues such as skin.

Solid tissue simulating phantoms having absorption at 970 nm for diffuse optics.

Tissue simulating phantoms can provide a valuable platform for quantitative evaluation of the performance of diffuse optical devices. While solid phantoms have been developed for applications related to characterizing exogenous fluorescence and intrinsic chromophores such as hemoglobin and melanin, we report the development of a poly(dimethylsiloxane) (PDMS) tissue phantom that mimics the spectral characteristics of tissue water. We have developed these phantoms to mimic different water fractions in tissue, with the purpose of testing new devices within the context of clinical applications such as burn wound triage. Compared to liquid phantoms, cured PDMS phantoms are easier to transport and use and have a longer usable life than gelatin-based phantoms. As silicone is hydrophobic, 9606 dye was used to mimic the optical absorption feature of water in the vicinity of 970 nm. Scattering properties are determined by adding titanium dioxide, which yields a wavelength-dependent scattering coefficient similar to that observed in tissue in the near-infrared. Phantom properties were characterized and validated using the techniques of inverse adding-doubling and spatial frequency domain imaging. Results presented here demonstrate that we can fabricate solid phantoms that can be used to simulate different water fractions

Low-cost tissue simulating phantoms with adjustable wavelength-dependent scattering properties in the visible and infrared ranges.

We present a method for low-cost fabrication of polydimethylsiloxane (PDMS) tissue simulating phantoms with tunable scattering spectra, spanning visible, and near-infrared regimes. These phantoms use optical polishing agents (aluminum oxide powders) at various grit sizes to approximate in vivo tissue scattering particles across multiple size distributions (range: 17 to 3 µm). This class of tunable scattering phantoms is used to mimic distinct changes in wavelength-dependent scattering properties observed in tissue pathologies such as partial thickness burns. Described by a power-law dependence on wavelength, the scattering magnitude of these phantoms scale linearly with particle concentration over a physiologic range [µs'=(0.5 to 2.0 mm−1)] whereas the scattering spectra, specific to each particle size distribution, correlate to distinct exponential coefficients (range: 0.007 to 0.32). Aluminum oxide powders used in this investigation did not detectably contribute to the absorption properties of these phantoms. The optical properties of these phantoms are verified through inverse adding-doubling methods and the tolerances of this fabrication method are discussed.

In vivo isolation of the effects of melanin from underlying hemodynamics across skin types using spatial frequency domain spectroscopy.

Skin is a highly structured tissue, raising concerns as to whether skin pigmentation due to epidermal melanin may confound accurate measurements of underlying hemodynamics. Using both venous and arterial cuff occlusions as a means of inducing differential hemodynamic perturbations, we present analyses of spectra limited to the visible or near-infrared regime, in addition to a layered model approach. The influence of melanin, spanning Fitzpatrick skin types I to V, on underlying estimations of hemodynamics in skin as interpreted by these spectral regions are assessed. The layered model provides minimal cross-talk between melanin and hemodynamics and enables removal of problematic correlations between measured tissue oxygenation estimates and skin phototype.

Optical properties of mouse brain tissue after optical clearing with FocusClear™.

Fluorescence microscopy is commonly used to investigate disease progression in biological tissues. Biological tissues, however, are strongly scattering in the visible wavelengths, limiting the application of fluorescence microscopy to superficial (<200µm) regions. Optical clearing, which involves incubation of the tissue in a chemical bath, reduces the optical scattering in tissue, resulting in increased tissue transparency and optical imaging depth. The goal of this study was to determine the time- and wavelength-resolved dynamics of the optical scattering properties of rodent brain after optical clearing with FocusClear™. Light transmittance and reflectance of 1-mm mouse brain sections were measured using an integrating sphere before and after optical clearing and the inverse adding doubling algorithm used to determine tissue optical scattering. The degree of optical clearing was quantified by calculating the optical clearing potential (OCP), and the effects of differing OCP were demonstrated using the optical histology method, which combines tissue optical clearing with optical imaging to visualize the microvasculature. We observed increased tissue transparency with longer optical clearing time and an analogous increase in OCP. Furthermore, OCP did not vary substantially between 400 and 1000 nm for increasing optical clearing durations, suggesting that optical histology can improve ex vivo visualization of several fluorescent probes.

In vivo measurements of cutaneous melanin across spatial scales: using multiphoton microscopy and spatial frequency domain spectroscopy.

The combined use of nonlinear optical microscopy and broadband reflectance techniques to assess melanin concentration and distribution thickness in vivo over the full range of Fitzpatrick skin types is presented. Twelve patients were measured using multiphoton microscopy (MPM) and spatial frequency domain spectroscopy (SFDS) on both dorsal forearm and volar arm, which are generally sun-exposed and non-sun-exposed areas, respectively. Both MPM and SFDS measured melanin volume fractions between (skin type I non-sun-exposed) and 20% (skin type VI sun exposed). MPM measured epidermal (anatomical) thickness values ~30-65 µm, while SFDS measured melanin distribution thickness based on diffuse optical path length. There was a strong correlation between melanin concentration and melanin distribution (epidermal) thickness measurements obtained using the two techniques. While SFDS does not have the ability to match the spatial resolution of MPM, this study demonstrates that melanin content as quantified using SFDS is linearly correlated with epidermal melanin as measured using MPM (R² = 0.8895). SFDS melanin distribution thickness is correlated to MPM values (R² = 0.8131). These techniques can be used individually and/or in combination to advance our understanding and guide therapies for pigmentation-related conditions as well as light-based treatments across a full range of skin types.

Quantitative short-wave infrared multispectral imaging of in vivo tissue optical properties.

Extending the wavelength range of spatial frequency domain imaging (SFDI) into the short-wave infrared (SWIR) has the potential to provide enhanced sensitivity to chromophores such as water and lipids that have prominent absorption features in the SWIR region. Here, we present, for the first time, a method combining SFDI with unstructured (zero spatial frequency) illumination to extract tissue absorption and scattering properties over a wavelength range (850 to 1800 nm) largely unexplored by previous tissue optics techniques. To obtain images over this wavelength range, we employ a SWIR camera in conjunction with an SFDI system. We use SFDI to obtain in vivo tissue reduced scattering coefficients at the wavelengths from 850 to 1050 nm, and then use unstructured wide-field illumination and an extrapolated power-law fit to this scattering spectrum to extract the absorption spectrum from 850 to 1800 nm. Our proof-of-principle experiment in a rat burn model illustrates that the combination of multispectral SWIR imaging, SFDI, and unstructured illumination can characterize in vivo changes in skin optical properties over a greatly expanded wavelength range. In the rat burn experiment, these changes (relative to normal, unburned skin) included increased absorption and increased scattering amplitude and slope, consistent with changes that we previously reported in the near-infrared using SFDI.

Polarization-sensitive hyperspectral imaging in vivo: a multimode dermoscope for skin analysis.

Attempts to understand the changes in the structure and physiology of human skin abnormalities by non-invasive optical imaging are aided by spectroscopic methods that quantify, at the molecular level, variations in tissue oxygenation and melanin distribution. However, current commercial and research systems to map hemoglobin and melanin do not correlate well with pathology for pigmented lesions or darker skin. We developed a multimode dermoscope that combines polarization and hyperspectral imaging with an efficient analytical model to map the distribution of specific skin bio-molecules. This corrects for the melanin-hemoglobin misestimation common to other systems, without resorting to complex and computationally intensive tissue optical models. For this system's proof of concept, human skin measurements on melanocytic nevus, vitiligo, and venous occlusion conditions were performed in volunteers. The resulting molecular distribution maps matched physiological and anatomical expectations, confirming a technologic approach that can be applied to next generation dermoscopes and having biological plausibility that is likely to appeal to dermatologists.

Quantitative near infrared spectroscopic analysis of Q-Switched Nd:YAG treatment of generalized argyria.

BACKGROUND AND OBJECTIVE: Generalized argyria is a blue-gray hyperpigmentation of the skin resulting from ingestion or application of silver compounds, such as silver colloid. Case reports have noted improvement after Q-Switched Neodymium-Yttrium Aluminum Garnet laser (1,064 nm QS Nd:YAG) laser treatment to small surface areas. No reports have objectively monitored laser treatment of generalized argyria over large areas of skin, nor have long-term outcomes been evaluated. STUDY DESIGN/MATERIALS AND METHODS: An incremental treatment plan was developed for a subject suffering from argyria. A quantitative near infrared spectroscopic measurement technique was employed to non-invasively analyze tissue-pigment characteristics pre- and post-laser treatment. Post-treatment measurements were collected at weeks 1, 2, 3, and 4, and again at 1 year. RESULTS: Immediate apparent removal of pigment was observed with 1 Q-switched 1,064 nm Nd:YAG laser treatment (3-6 mm spot; 0.8-2 J/cm(2) ) per area. Entire face, neck, upper chest, and arms were treated over multiple sessions. Treatments were very painful and general anesthesia was utilized in order to treat large areas. Near-infrared spectroscopy was used to characterize and quantify the concentration of silver particles in the dermis based on the absorption features of the silver particles as well as the optical scattering effects they impart. We were able to estimate that there was, on average, 0.042 mg/ml concentration of silver prior to treatment and that these levels went below the minimum detectable limit of the instrument post-treatment. There was no recurrence of discoloration over the 1-year study period. CONCLUSION: QS 1,064 nm laser treatment of argyria is a viable method to restore normal skin pigmentation with no evidence of recurrence over study period. Quantitative spectroscopic measurements: (1) confirmed dyspigmentation was due to silver, (2) validated our clinical assessment of no recurrence up to 1-year post-treatment, and (3) indicated no collateral tissue damage with treatments.