Collagen ultrastructural symmetry and its malignant alterations in human breast cancer revealed by polarization‐resolved second‐harmonic generation microscopy

Several specific alterations of the extracellular matrix can be considered a distinctive hallmark of cancer. In particular, a different morphology of the collagen scaffold is frequently found within the peritumoural environment. In this study, we report about a significant difference in the ultrastructural organization of collagen at the supra‐molecular level between the perilesional scaffold and the tumour area in human breast carcinoma samples. In particular, we demonstrated that polarization‐resolved second‐harmonic generation (P‐SHG) microscopy is able to link the altered collagen architecture at the ultrastructural level found in perilesional tissue with a different organization of collagen fibrils at the molecular level.     

Exploring the utility of Deep Red Anthraquinone 5 for digital staining of ex vivo confocal micrographs of optically sectioned skin

We investigated the utility of the fluorescent dye Deep Red Anthraquinone 5 (DRAQ5) for digital staining of optically sectioned skin in comparison to acridine orange (AO). Eight fresh-frozen thawed Mohs discard tissue specimens were stained with AO and DRAQ5, and imaged using an ex vivo confocal microscope at three wavelengths (488 nm and 638 nm for fluorescence, 785 nm for reflectance). Images were overlaid (AO + Reflectance, DRAQ5 + Reflectance), digitally stained, and evaluated by three investigators for perceived image quality (PIQ) and histopathological feature identification. In addition to nuclear staining, AO seemed to stain dermal fibers in a subset of cases in digitally stained images, while DRAQ5 staining was more specific to nuclei. Blinded evaluation showed substantial agreement, favoring DRAQ5 for PIQ (82%, Cl 75%-90%, Gwet's AC 0.74) and for visualization of histopathological features in (81%, Cl 73%-89%, Gwet's AC 0.67), supporting its use in digital staining of multimodal confocal micrographs of skin.     

Integrated photoacoustic and hyperspectral dual‐modality microscopy for co‐imaging of melanoma and cutaneous squamous cell carcinoma in vivo

Skin carcinoma such as melanoma (MM) and cutaneous squamous cell carcinoma (cSCC) are considered as the highest mortality and the most aggressive skin cancers in dermatology. In view that early diagnosis and treatment can greatly improve the survival rate and life quality of the patients, developing noninvasive and effective evaluation methods is of great significance for the detection and identification of early stage cutaneous cancers. In this article, we propose a hybrid photoacoustic and hyperspectral dual‐modality microscopy to evaluate and differentiate skin carcinoma by structural and multiphysiological parameters. The proposed system's imaging abilities are verified by mimic phantoms and normal mice experiments. Furthermore, in vivo characterization and evaluation results of MM and cSCC mice are obtained successfully, which prove this novel method could be used as a reliable and useful method for skin cancer detection in early stages.     

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.     

Intuitive and broadly applicable definitions of niche and fitness differences

Explaining nature’s biodiversity is a key challenge for science. To persist, populations must be able to grow faster when rare, a feature called negative frequency dependence and quantified as ‘niche differences’ () in modern coexistence theory. Here, we first show that available definitions of differ in how link to species interactions, are difficult to interpret and often apply to specific community types only. We then present a new definition of that is intuitive and applicable to a broader set of (modelled and empirical) communities than is currently the case, filling a main gap in the literature. Given , we also redefine fitness differences () and illustrate how and determine coexistence. Finally, we demonstrate how to apply our definitions to theoretical models and experimental data, and provide ideas on how they can facilitate comparison and synthesis in community ecology.     

Imaging sebaceous gland using optical coherence tomography with deep learning assisted automatic identification

Imaging sebaceous glands and evaluating morphometric parameters are important for diagnosis and treatment of serum problems. In this article, we investigate the feasibility of high-resolution optical coherence tomography (OCT) in combination with deep learning assisted automatic identification for these purposes. Specifically, with a spatial resolution of 2.3 μm × 6.2 μm (axial × lateral, in air), OCT is capable of clearly differentiating sebaceous gland from other skin structures and resolving the sebocyte layer. In order to achieve efficient and timely imaging analysis, a deep learning approach built upon ResNet18 is developed to automatically classify OCT images (with/without sebaceous gland), with a classification accuracy of 97.9%. Based on the result of automatic identification, we further demonstrate the possibility to measure gland size, sebocyte layer thickness and gland density.     

A new strategy for canine visceral leishmaniasis diagnosis based on FTIR spectroscopy and machine learning

Visceral leishmaniasis is a neglected disease caused by protozoan parasites of the genus Leishmania. The successful control of the disease depends on its accurate and early diagnosis, which is usually made by combining clinical symptoms with laboratory tests such as serological, parasitological, and molecular tests. However, early diagnosis based on serological tests may exhibit low accuracy due to lack of specificity caused by cross-reactivities with other pathogens, and sensitivity issues related, among other reasons, to disease stage, leading to misdiagnosis. In this study was investigated the use of mid-infrared spectroscopy and multivariate analysis to perform a fast, accurate, and easy canine visceral leishmaniasis diagnosis. Canine blood sera of 20 noninfected, 20 Leishmania infantum, and eight Trypanosoma evansi infected dogs were studied. The data demonstrate that principal component analysis with machine learning algorithms achieved an overall accuracy above 85% in the diagnosis.     

Determination of the diameter of simulated human capillaries using shifted position-diffuse reflectance imaging

Multiple diseases are associated with a wide spectrum of microvascular dysfunctions, microangiopathies and microcirculation disorders. Monitoring the microcirculation could thus be useful to diagnose many local and systemic circulatory disorders and to supervise critically ill patients. Many of the scores currently available to help identify the condition of a microcirculation disorder are invasive or leave scope for interpretation. Thus, the present study aims to investigate with Monte-Carlo simulations (as numerical solutions of the radiative transfer equation) whether shifted position-diffuse reflectance imaging (SP-DRI), a non-invasive diagnostic technique, reveals information on the capillary diameter to assess the state of the microcirculation. To quantify the SP-DRI signal, the modulation parameter K is introduced. It proves to correlate almost perfectly with the capillary diameter (), making it a valid parameter for reliably assessing microcirculation. SP-DRI is emerging as an important milestone on the way to early and conveniently diagnosing microcirculation associated diseases.     

Clinical applications of non-invasive multi and hyperspectral imaging of cell and tissue autofluorescence beyond oncology

Hyperspectral and multispectral imaging of cell and tissue autofluorescence employs fluorescence imaging, without exogenous fluorophores, across multiple excitation/emission combinations (spectral channels). This produces an image stack where each pixel (matched by location) contains unique information about the sample's spectral properties. Analysis of this data enables access to a rich, molecularly specific data set from a broad range of cell-native fluorophores (autofluorophores) directly reflective of biochemical status, without use of fixation or stains. This non-invasive, non-destructive technology has great potential to spare the collection of biopsies from sensitive regions. As both staining and biopsy may be impossible, or undesirable, depending on the context, this technology great diagnostic potential for clinical decision making. The main research focus has been on the identification of neoplastic tissues. However, advances have been made in diverse applications—including ophthalmology, cardiovascular health, neurology, infection, assisted reproduction technology and organ transplantation.     

Inside Cover: Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

A type of compact and cost‐effective light‐sheet imaging device, termed sub‐voxel‐resolving light‐sheet add‐on module (SLAM), is developed to cooperate with conventional 2D epifluorescence microscope, allowing high‐contrast, resolution‐improved 3D imaging of various biological samples at high throughput. Further details can be found in the article by Fang Zhao, Yicong Yang, Yi Li, et al. ( e201960243 ).

     

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

     

Evidence for Depressed Reproduction of Golden Eagles in Washington

Beginning in 1977 the Washington Department of Fish and Wildlife conducted annual surveys to determine statewide golden eagle (Aquila chrysaetos) occupancy and productivity. Current interest in the regional and national status of the species prompted our investigation to determine utility of historical data in assessing trends in reproduction, and to test efficacy of a sampling protocol that surveyed randomly selected territories and also accounted for detection probability. We found evidence indicating poor reproduction from 38 annual surveys conducted at 301 known territories statewide between 1977 and 2014. At 256 territories in eastern Washington, USA, apparent occupancy was low ( = 50.9%) and nesting success declined by 22%. All reproductive parameters were higher than at 45 territories in western Washington. We tested efficacy of a sampling protocol in 2013 and 2014 by surveying 108 randomly selected eastern territories. Probability of detecting eagles for these years from ground (= 89%) was greater than from air (= 66%). Our estimate of territory occupancy, corrected by probability of detection, was lower in 2013 (= 56.7%, 95% CI = 46.3–66.7%) than in 2014 (= 73.7%, 95% CI = 64.8–81.7%), as was the estimated number of breeding pairs (2013: = 158, 95% CI = 151–164; 2014: = 187, 95% CI = 182–192). Higher productivity (young/occupied territory) in 2013 (= 0.59, 95% CI = 0.40–0.82) than in 2014 (= 0.41, 95% CI = 0.27–0.59) and lower proportions of ≥1 immature eagle among nesting pairs in 2013 (16%) than in 2014 (31%), suggested higher immature pairing among sampled pairs contributed to inter-year differences in these reproductive parameters. Current and historical evidence for depressed golden eagle nesting in Washington is consistent with documented effects from habitat conversion, prey declines, lead contamination, and wind power development. We recommend future surveys in eastern Washington adhere to the random sampling protocol and conduct surveys at regular intervals to allow for trend analysis of reproductive parameters to better monitor golden eagle status. Surveys in western Washington, conducted exclusively from ground at all nests, will improve detection and cost efficiency. © 2020 The Wildlife Society.     

Multimodal data acquisition at SARS-CoV-2 drive through screening centers: Setup description and experiences in Saarland,Germany

SARS-CoV-2 drive through screening centers (DTSC) have been implemented worldwide as a fast and secure way of mass screening. We use DTSCs as a platform for the acquisition of multimodal datasets that are needed for the development of remote screening methods. Our acquisition setup consists of an array of thermal, infrared and RGB cameras as well as microphones and we apply methods from computer vision and computer audition for the contactless estimation of physiological parameters. We have recorded a multimodal dataset of DTSC participants in Germany for the development of remote screening methods and symptom identification. Acquisition in the early stages of a pandemic and in regions with high infection rates can facilitate and speed up the identification of infection specific symptoms and large-scale data acquisition at DTSC is possible without disturbing the flow of operation.     

The effects of hurricanes on the stochastic population growth of the endemic epiphytic orchid Broughtonia cubensis living in Cuba

We carried out a posthurricane evaluation of Broughtonia cubensis (Lindl.) Cogn., an endemic Cuban epiphytic orchid, after Hurricane Ivan (2004). We studied the transient responses in the stochastic dynamics of the species at three different sites over 13 successive years (2006–2019), monitored plot inventories (464 individuals in 10 transects) and built stochastic population models. The deterministic stochastic growth rate values () did not significantly differ (F = 2.76; p > 0.076) among the three sites over the 2006–2019 period. The long-term stochastic growth rate was 0.973 [0.932, 1.034]. The matrix elements that had the largest effect on were the transition to and stasis within the largest size class. Transient responses explained an average of 86% of the variation in the observed population growth rates , compared to 4% of the variation in the vital rates . Because transient dynamics are dependent on the population size composition, we ran extinction risk analyses under two scenarios: a population composed mainly of juveniles and another composed mainly of adults. There was little risk of falling below the quasi-extinction threshold before 25 year for both juveniles and adults. However, the risk of quasi-extinction was almost certain for both size classes by 80 year. We also simulated the effect of increasing the hurricane occurrence probability over 80 year on the population. There was little risk of extinction before 20 year in the baseline model, but there was a significant risk of extinction within 5 year when 90% of the individuals were affected by a new hurricane event.     

Fluorescence visualization of the neuropathic pain triad in trigeminal neuralgia

Trigeminal neuralgia (TN), an exemplary condition of neuropathic facial pain, seriously affects the physical and mental health of patients, becoming a major medical and social problem. So far, the mechanism of TN and its relation to neuronal activity remain unclear, largely limited by the spatial resolution of visualization methods. In the meanwhile, current therapeutic strategies targeting neurons have not achieved satisfactory outcome. Here, we investigate the neuropathic pain triad in TN by establishing an animal model of TN by chronic constriction injury of the unilateral infraorbital nerve (ION-CCI) and leveraging the single-cell resolution of confocal microscopy, including neuronal hyperexcitability, glial activation, and macrophage polarization. These results can broaden the understanding of TN pathogenesis from neurons to the neuropathic pain triad, and suggest that optical microscopy can provide new opportunities for understanding the complex pathogenesis of TN at single-cell resolution, potentially contributing to the identification of more precise therapeutic targets and the development of more effective treatment modalities.     

Estimating the decision curve and its precision from three study designs

The decision curve plots the net benefit of a risk model for making decisions over a range of risk thresholds, corresponding to different ratios of misclassification costs. We discuss three methods to estimate the decision curve, together with corresponding methods of inference and methods to compare two risk models at a given risk threshold. One method uses risks (R) and a binary event indicator (Y) on the entire validation cohort. This method makes no assumptions on how well-calibrated the risk model is nor on the incidence of disease in the population and is comparatively robust to model miscalibration. If one assumes that the model is well-calibrated, one can compute a much more precise estimate of based on risks R alone. However, if the risk model is miscalibrated, serious bias can result. Case–control data can also be used to estimate if the incidence (or prevalence) of the event () is known. This strategy has comparable efficiency to using the full data, and its efficiency is only modestly less than that for the full data if the incidence is estimated from the mean of Y. We estimate variances using influence functions and propose a bootstrap procedure to obtain simultaneous confidence bands around the decision curve for a range of thresholds. The influence function approach to estimate variances can also be applied to cohorts derived from complex survey samples instead of simple random samples.     

Pathological crystal imaging with single‐shot computational polarized light microscopy

Pathological crystal identification is routinely practiced in rheumatology for diagnosing arthritis disease such as gout, and relies on polarized light microscopy as the gold standard method used by medical professionals. Here, we present a single‐shot computational polarized light microscopy method that reconstructs the transmittance, retardance and slow‐axis orientation of a birefringent sample using a single image captured with a pixelated‐polarizer camera. This method is fast, simple‐to‐operate and compatible with all the existing standard microscopes without extensive or costly modifications. We demonstrated the success of our method by imaging three different types of crystals found in synovial fluid and reconstructed the birefringence information of these samples using a single image, without being affected by the orientation of individual crystals within the sample field‐of‐view. We believe this technique will provide improved sensitivity, specificity and speed, all at low cost, for clinical diagnosis of crystals found in synovial fluid and other bodily fluids.     

Issue Information

Cover Photograph: Upper left: Simultaneous induction of ureagenesis and gluconeogenesis coincides with a corticosterone surge occurring prior to metamorphosis in Xenopus laevis (see Konno, pp. 6–15). Upper right: Transverse sections of the tibiofibula at Gosner stage 42 (top panels), of Xenopus tropicalis (left) and Ceratophrys cranwelli (right); Ca: cartilage, CB: cortical bone, Mu: muscle. Bottom panels show adult frogs (see Kondo et al. pp. 16–22). Lower left: Maea knockdown interferes with head formation. Wild type (top) and morphant phenotypes with small eyes (middle) and no eyes (bottom) are shown (see Goto et al. pp. 29–36). Lower right: ChIP-seq analyses of target genes of thyroid hormone receptors (TR) in tail, hindlimb and intestine in tadpoles during Xenopus metamorphosis. Venn diagrams show much fewer TR-bound genes in TRa-knockout X. tropicalis frogs compared to wild type (see Tanizaki et al. pp. 23–28).

     

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.

     

Specific labeling and identification of bacteria based on concentration-dependent carbon dot staining combined with hyperspectral imaging

Concentration-dependent carbon dot (CD) fluorescence was developed and utilized alongside hyperspectral microscopy as a specific labeling and identification technique for bacteria. Staining revealed that the CD concentration within cells depended on the characteristic intracellular environment of the species. Therefore, based on the concentration dependence of the CD fluorescence, different bacterial species were specifically labeled. Hyperspectral microscopy captured subtle fluorescence variations to identify bacteria. Method validation using Bacillus subtilis and Bacillus licheniformis succeeded with an identification accuracy of 99%. As a simple, rapid method for labeling and identifying bacterial species in mixtures, this technique has excellent potential for bacterial community studies.