Optical investigation of three‐dimensional human skin equivalents: A pilot study

Human skin equivalents (HSEs) are three‐dimensional living models of human skin that are prepared in vitro by seeding cells onto an appropriate scaffold. They recreate the structure and biological behaviour of real skin, allowing the investigation of processes such as keratinocyte differentiation and interactions between the dermal and epidermal layers. However, for wider applications, their optical and mechanical properties should also replicate those of real skin. We therefore conducted a pilot study to investigate the optical properties of HSEs. We compared Monte Carlo simulations of (a) real human skin and (b) two‐layer optical models of HSEs with (c) experimental measurements of transmittance through HSE samples. The skin layers were described using a hybrid collection of optical attenuation coefficients. A linear relationship was observed between the simulations and experiments. For samples thinner than 0.5 mm, an exponential increase in detected power was observed due to fewer instances of absorption and scattering.      

Inside Cover: Comparative analysis of intrinsic skin aging between Caucasian and Asian subjects by slide‐free in vivo harmonic generation microscopy (J. Biophotonics 4/2020)

A comparative analysis of intrinsic skin aging between Caucasian and Asian subjects by slide‐free in vivo harmonic generation microscopy is reported. The cellular and nuclear areas of basal cells in Caucasians and Asians are found to increase at the same rate, ideal for scoring age. On the other hand, the viable epidermis thickness, the dermal papilla volume and height in Caucasians are found to decrease faster than those in Asians. Further details can be found in the article by Kuan‐Hung Lin, Yi‐Hua Liao, Ming‐Liang Wei, and Chi‐Kuang Sun ( e201960063 ).

     

Polarization-resolved SHG imaging as a fast screening method for collagen alterations during aging: Comparison with light and electron microscopy

Our previous study on rat skin showed that cumulative oxidative pressure induces profound structural and ultrastructural alterations in both rat skin epidermis and dermis during aging. Here, we aimed to investigate the biophotonic properties of collagen as a main dermal component in the function of chronological aging. We used second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) on 5 μm thick skin paraffin sections from 15-day-, 1-month- and 21-month-old rats, respectively, to analyze collagen alterations, in comparison to conventional light and electron microscopy methods. Obtained results show that polarization-resolved SHG (PSHG) images can detect collagen fiber alterations in line with chronological aging and that this method is consistent with light and electron microscopy. Moreover, the β coefficient calculated from PSHG images points out that delicate alterations lead to a more ordered structure of collagen molecules due to oxidative damage. The results of this study also open the possibility of successfully applying this fast and label-free method to previously fixed samples.     

Dual‐ and single‐shot susceptibility ratio measurements with circular polarizations in second‐harmonic generation microscopy

Polarization‐resolved second‐harmonic generation (P‐SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio , with z‐axis parallel and x‐axis perpendicular to the C₆ symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P‐SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of : A dual‐shot configuration where the SHG circular anisotropy generated using incident right‐ and left‐handed circularly‐polarized light is measured; and a single‐shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes‐Mueller polarimetry. The dual‐ and single‐shot circular anisotropy measurements can be used for fast imaging that is independent of the in‐plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas.     

Issue Information

Cover Image An infl orescence of Clerodendrum izuinsulare (left) and C. trichotomum (right). Photos by Natsuki Aihara in Niijima Island (left) and by Takashi Miyake in Izu Peninsula (right), Japan. Miyake et al., doi.org/10.1111/1442-1984.12287

     

Multispectral near infrared absorption imaging for histology of skin cancer

Multispectral imaging combines the spectral resolution of spectroscopy with the spatial resolution of imaging and is therefore very useful for biomedical applications. Currently, histological diagnostics use mainly stainings with standard dyes (eg, hematoxylin + eosin) to identify tumors. This method is not applicable in vivo and provides low amounts of chemical information. Biomolecules absorb near infrared light (NIR, 800‐1700 nm) at different wavelengths, which could be used to fingerprint tissue. Here, we built a NIR multispectral absorption imaging setup to study skin tissue samples. NIR light (900‐1500 nm) was used for homogenous wide‐field transmission illumination and detected by a cooled InGaAs camera. In this setup, images I(x, y, λ) from dermatological samples (melanoma, nodular basal‐cell carcinoma, squamous‐cell carcinoma) were acquired to distinguish healthy from diseased tissue regions. In summary, we show the potential of multispectral NIR imaging for cancer diagnostics.      

Determination and correction of aberrations in full field optical coherence tomography using phase gradient autofocus by maximizing the likelihood function

A method for numerical estimation and correction of aberrations of the eye in fundus imaging with optical coherence tomography (OCT) is presented. Aberrations are determined statistically by using the estimate based on likelihood function maximization. The method can be considered as an extension of the phase gradient autofocusing algorithm in synthetic aperture radar imaging to 2D optical aberration correction. The efficacy of the proposed method has been demonstrated in OCT fundus imaging with 6λ aberrations. After correction, single photoreceptors were resolved. It is also shown that wave front distortions with high spatial frequencies can be determined and corrected.     

Isotopically anomalous organic carbon in the aftermath of the Marinoan snowball Earth

Throughout most of the sedimentary record, the marine carbon cycle is interpreted as being in isotopic steady state. This is most commonly inferred via isotopic reconstructions, where two export fluxes (organic carbon and carbonate) are offset by a constant isotopic fractionation of ~25 (termed ). Sedimentary deposits immediately overlying the Marinoan snowball Earth diamictites, however, stray from this prediction. In stratigraphic sections from the Ol Formation (Mongolia) and Sheepbed Formation (Canada), we observe a temporary excursion where the organic matter has anomalously heavy C and is grossly decoupled from the carbonate C. This signal may reflect the unique biogeochemical conditions that persisted in the aftermath of snowball Earth. For example, physical oceanographic modeling suggests that a strong density gradient caused the ocean to remain stratified for about 50,000 years after termination of the Marinoan snowball event, during which time the surface ocean and continental weathering consumed the large atmospheric CO₂ reservoir. Further, we now better understand how C records of carbonate can be post‐depostionally altered and thus be misleading. In an attempt to explain the observed carbon isotope record, we developed a model that tracks the fluxes and isotopic values of carbon between the surface ocean, deep ocean, and atmosphere. By comparing the model output to the sedimentary data, stratification alone cannot generate the anomalous observed isotopic signal. Reproducing the heavy C in organic matter requires the progressively diminishing contribution of an additional anomalous source of organic matter. The exact source of this organic matter is unclear.     

Translational optical coherence elastography for assessment of systemic sclerosis

Systemic sclerosis (SSc‐scleroderma) is an autoimmune disorder with high mortality rate that results in excessive accumulation of collagen in the skin and internal organs. Currently, the modified Rodnan Skin Score (mRSS) is the gold standard for evaluating the dermal thickening due to SSc. However, mRSS has noticeable inter‐ and intra‐observer variabilities as quantified by the interclass correlation coefficient (ICC: 0.6‐0.75). In this work, optical coherence elastography (OCE) combined with structural optical coherence tomography (OCT) image analysis was used to assess skin thickness in 12 SSc patients and healthy volunteers. Inter‐ (ICC: 0.62‐0.99) and intra‐observer (ICC > 0.90) assessment of OCT/OCE showed excellent reliability. Clinical assessments, including histologically assessed dermal thickness (DT), mRSS, and site‐specific mRSS (SMRSS) were also performed for further validation. The OCE and OCT results from the forearm demonstrated the highest correlation (OCE: 0.78, OCT: 0.65) with SMRSS. Importantly, OCE and OCT had stronger correlations with the histological DT (OCT: r = .78 and OCE: r = .74) than SMRSS (r = .57), indicating the OCT/OCE could outperform semi‐quantitative clinical assessments such as SMRSS. Overall, these results demonstrate that OCT/OCE could be useful for rapid, noninvasive and objective assessments of SSc onset and monitoring skin disease progression and treatment response.     

Issue Information

Jackson County, Illinois, U.S.A., Photo by Nathan Soley (right) Champaign County, Illinois, U.S.A., Photo by Nathan Soley (left) Soley and Sipes “Reproductive biology and pollinators of the invasive shrub Autumn olive (Elaeagnus umbellata Thunberg)”. https://doi.org/10.1111/1442-1984.12307

     

Respirometer in a box: development and use of a portable field respirometer for estimating oxygen consumption of large-bodied fishes

This study developed a portable, low-cost field respirometer for measuring oxygen consumption rates of large-bodied fishes. The respirometer performed well in laboratory tests and was used to measure the oxygen consumption rates ( O₂) of bull sharks Carcharhinus leucas (mean: 249.21 ± 58.10 mg O₂ kg⁻¹ h⁻¹ at 27.05°C). Interspecific comparisons and assessments of oxygen degradation curves indicated that the respirometer provided reliable measurements of O₂. This system presents a field-based alternative to laboratory respirometers, opening opportunities for studies on species in remote localities, increasing the ability to validate physiological field studies.     

Impact of optical clearing on ex vivo human skin optical properties characterized by spatially resolved multimodal spectroscopy

A spatially resolved multimodal spectroscopic device was used on a two-layered “hybrid” model made of ex vivo skin and fluorescent gel to investigate the effect of skin optical clearing on the depth sensitivity of optical spectroscopy. Time kinetics of fluorescence and diffuse reflectance spectra were acquired in four experimental conditions: with optical clearing agent (OCA) 1 made of polyethylene glycol 400 (PEG-400), propylene glycol and sucrose; with OCA 2 made of PEG-400 and dimethyl sulfoxide (DMSO); with saline solution as control and a “dry” condition. An increase in the gel fluorescence back reflected intensity was measured after optical clearing. Effect of OCA 2 turned out to be stronger than that of OCA 1, possibly due to DMSO impact on the stratum corneum keratin conformation. Complementary experimental results showed increased light transmittance through the skin and confirmed that the improvement in the depth sensitivity of the multimodal spectroscopic approach is related not only to the dehydration and refractive indices matching due to optical clearing, but also to the mechanical compression of tissues caused by the application of the spectroscopic probe.     

Issue Information

Left panel: Lateral view of a lenticular cell of Valonia utricularis at the beginning (upper) and the end (lower) of a 22-hr observation. Arrowheads with numbers indicate carbon particles along the cell outline. Right panel: (upper) Changes in cell outline and position of the numbered carbon particles in the lenticular cell at 2-hr intervals. (lower) The segmental extension profile of the lenticular cell showing the anisotropic cell surface growth. Numbers represent those used for labelling the carbon particles. See Mine et al. in this issue for details.

     

Another chemolithotrophic metabolism missing in nature: sulfur comproportionation

Chemotrophic microorganisms gain energy for cellular functions by catalyzing oxidation–reduction (redox) reactions that are out of equilibrium. Calculations of the Gibbs energy ( ΔG _r ) can identify whether a reaction is thermodynamically favourable and quantify the accompanying energy yield at the temperature, pressure and chemical composition in the system of interest. Based on carefully calculated values of ΔG _r , we predict a novel microbial metabolism – sulfur comproportionation (3H₂S + + 2H⁺ ⇌ 4S⁰ + 4H₂O). We show that at elevated concentrations of sulfide and sulfate in acidic environments over a broad temperature range, this putative metabolism can be exergonic ( ΔG _r <0), yielding ~30–50 kJ mol⁻¹. We suggest that this may be sufficient energy to support a chemolithotrophic metabolism currently missing from the literature. Other versions of this metabolism, comproportionation to thiosulfate (H₂S + ⇌ + H₂O) and to sulfite (H₂S + 3 ⇌ 4 + 2H⁺), are only moderately exergonic or endergonic even at ideal geochemical conditions. Natural and impacted environments, including sulfidic karst systems, shallow-sea hydrothermal vents, sites of acid mine drainage, and acid–sulfate crater lakes, may be ideal hunting grounds for finding microbial sulfur comproportionators.     

Automatic imaging of Drosophila embryos with light sheet fluorescence microscopy on chip

We present a microscope on chip for automated imaging of Drosophila embryos by light sheet fluorescence microscopy. This integrated device, constituted by both optical and microfluidic components, allows the automatic acquisition of a 3D stack of images for specimens diluted in a liquid suspension. The device has been fully optimized to address the challenges related to the specimens under investigation. Indeed, the thickness and the high ellipticity of Drosophila embryos can degrade the image quality. In this regard, optical and fluidic optimization has been carried out to implement dual-sided illumination and automatic sample orientation. In addition, we highlight the dual color investigation capabilities of this device, by processing two sample populations encoding different fluorescent proteins. This work was made possible by the versatility of the used fabrication technique, femtosecond laser micromachining, which allows straightforward fabrication of both optical and fluidic components in glass substrates.     

Issue Information

Cover Image The pistillate-phase flowers of Artabotrys (Annonaceae): A. blumei (left) and A. brachypetalus (right). Photos by Junhao Chen, taken in Hong Kong and South Africa, respectively (DOI: 10.1111/1442-1984.12273 ).

     

A new deep learning method for image deblurring in optical microscopic systems

Deconvolution is the most commonly used image processing method in optical imaging systems to remove the blur caused by the point‐spread function (PSF). While this method has been successful in deblurring, it suffers from several disadvantages, such as slow processing time due to multiple iterations required to deblur and suboptimal in cases where the experimental operator chosen to represent PSF is not optimal. In this paper, we present a deep‐learning‐based deblurring method that is fast and applicable to optical microscopic imaging systems. We tested the robustness of proposed deblurring method on the publicly available data, simulated data and experimental data (including 2D optical microscopic data and 3D photoacoustic microscopic data), which all showed much improved deblurred results compared to deconvolution. We compared our results against several existing deconvolution methods. Our results are better than conventional techniques and do not require multiple iterations or pre‐determined experimental operator. Our method has several advantages including simple operation, short time to compute, good deblur results and wide application in all types of optical microscopic imaging systems. The deep learning approach opens up a new path for deblurring and can be applied in various biomedical imaging fields.     

A new brain-penetrant glucosylceramide synthase inhibitor as potential Therapeutics for Gaucher disease

Gaucher disease (GD), the most common lysosomal storage disorders, is caused by GBA gene mutations resulting in glycosphingolipids accumulations in various tissues, such as the brain. While suppressing glycosphingolipid accumulation is the central strategy for treating peripheral symptoms of GD, there is no effective treatment for the central nervous system symptoms. As glycosphingolipid biosynthesis starts from ceramide glycosylation by glucosylceramide synthase (GCS), inhibiting GCS in the brain is a promising strategy for neurological GD. Herein, we discovered T-036, a potent and brain-penetrant GCS inhibitor with a unique chemical structure and binding property. T-036 does not harbor an aliphatic amine moiety and has a noncompetitive inhibition mode to the substrates, unlike other known inhibitors. T-036 exhibited sufficient exposure and a significant reduction of glucosylsphingolipids in the plasma and brain of the GD mouse model. Therefore, T-036 could be a promising lead molecule for treating central nervous system symptoms of GD.

     

Issue Information

CRISPR-Cas9 system with PEG-mediated transfection was efficient for genome editing in Ulva prolifera. U. prolifera (left) and male gametes (middle). Wild type (upper right) and the genome-edited strain (lower right) cultured in 2-FA selection medium, and each mutation site on the APT gene. See Ichihara et al. in this issue.

     

Joint time-frequency analysis of visible laser reflections in a sheep heart

Limited methods exist to confirm the position of cardiovascular devices in the superior vena cava or right atrium of the heart. The aim of this study was to design, test and validate the feasibility of whether an optical fiber-based instrument could accurately distinguish when a cardiovascular catheter was located in the superior vena cava vs in the right atrium. An optical fiber was placed in a cardiovascular catheter which was inserted into a living sheep and guided to the vicinity of the heart where diode laser-based reflection intensity data were simultaneously gathered from two visible wavelengths of light reflected from the venous and atrial tissue surfaces near the cavoatrial junction. The time series data were postoperatively analyzed using methods of joint time-frequency analysis and validated against catheter positions determined with fluoroscopy and ECG. The system was successful in distinguishing the location of the superior vena cava from the right atrium.