Physicochemical characteristics of airborne microplastics of a typical coastal city in the Yangtze River Delta Region, China.

Airborne microplastics (MPs) are important pollutants that have been present in the environment for many years and are characterized by their universality, persistence, and potential toxicity. This study investigated the effects of terrestrial and marine transport of MPs in the atmosphere of a coastal city and compared the difference between daytime and nighttime. Laser direct infrared imaging (LDIR) and polarized light microscopy were used to characterize the physical and chemical properties of MPs, including number concentration, chemical types, shape, and size. Backward trajectories were used to distinguish the air masses from marine and terrestrial transport. Twenty chemical types were detected by LDIR, with rubber (16.7%) and phenol-formaldehyde resin (PFR; 14.8%) being major components. Three main morphological types of MPs were identified, and fragments (78.1%) are the dominant type. MPs in the atmosphere were concentrated in the small particle size segment (20-50 µm). The concentration of MPs in the air mass from marine transport was 14.7 items/m3 - lower than that from terrestrial transport (32.0 items/m3). The number concentration of airborne MPs was negatively correlated with relative humidity. MPs from terrestrial transport were mainly rubber (20.2%), while those from marine transport were mainly PFR (18%). MPs in the marine transport air mass were more aged and had a lower number concentration than those in the terrestrial transport air mass. The number concentration of airborne MPs is higher during the day than at night. These findings could contribute to the development of targeted control measures and methods to reduce MP pollution.

Mineralogical investigation of asbestos contamination of soil near old vermiculite processing plant in Honolulu, Hawai'i.

From 1954 to 1983, a vermiculite processing facility operated near the Honolulu airport and processed raw material from the Libby, Montana mine, which is now well known for the high asbestos content of its clay deposits. The factory was closed in 1983 due to health hazard concerns, and remediation was performed in 2001 as part of the Libby mine superfund project. However, because of close proximity of the closed-down facility to residential areas of metropolitan Honolulu, some concerns remain regarding the possible environmental persistence of the harmful contaminant. To assess the dispersion of asbestos-contaminated vermiculite and explore the impact of trade winds on its distribution, air samples, and soil samples were collected from multiple locations near the former vermiculite plant. Polarized light microscopy was employed to identify elongated minerals, including potential asbestos. Quantitative mineralogical analysis utilizing X-ray powder diffraction and Rietveld refinement revealed an average content of approximately 7% vermiculite and 4% tremolite at the site. The asbestiform nature of tremolite was confirmed through X-ray micro-diffraction. Detailed analysis of airborne samples using transmission electron microscopy revealed no detectable levels of asbestos fibers in the vicinity of the former processing facilities, but the possibility of asbestos fibers becoming airborne due to mechanical disturbance during dry weather cannot be ruled out.

Ex ovo omnia-why don't we know more about egg quality via imaging?

Determining egg quality is the foremost challenge in assisted reproductive technology (ART). Although extensive advances have been made in multiple areas of ART over the last 40 years, oocyte quality assessment tools have not much evolved beyond standard morphological observation. The oocyte not only delivers half of the nuclear genetic material and all of the mitochondrial DNA to an embryo but also provides complete developmental support during embryonic growth. Oocyte mitochondrial numbers far exceed those of any somatic cell, yet little work has been done to evaluate the mitochondrial bioenergetics of an oocyte. Current standard oocyte assessment in in vitro fertilization (IVF) centers include the observation of oocytes and their surrounding cell complex (cumulus cells) via stereomicroscope or inverted microscope, which is largely primitive. Additional oocyte assessments include polar body grading and polarized light meiotic spindle imaging. However, the evidence regarding the aforementioned methods of oocyte quality assessment and IVF outcomes is contradictory and non-reproducible. High-resolution microscopy techniques have also been implemented in animal and human models with promising outcomes. The current era of oocyte imaging continues to evolve with discoveries in artificial intelligence models of oocyte morphology selection albeit at a slow rate. In this review, the past, current, and future oocyte imaging techniques will be examined with the goal of drawing attention to the gap which limits our ability to assess oocytes in real time. The implications of improved oocyte imaging techniques on patients undergoing IVF will be discussed as well as the need to develop point of care oocyte assessment testing in IVF labs.

On the structural and mechanical properties of mixed coconut and olive oil oleogels and bigels.

The interaction of monoglycerides and phytosterols in olive- and coconut oil on the structuring of oleogels was analyzed. Specifically, bigels with gelatin hydrogel in different ratios (40:60 and 60:40 w/w) were formed. The physicochemical and microstructural attributes of these systems were assessed. The olive oil to coconut oil ratio (0-100 w/w) and the added oleogelators affected the crystal structure and the mechanical properties of the oleogels. Polarized light microscopy revealed that the addition of coconut oil created a denser triglycerides crystal network and the presence of phytosterols created more needle-like crystals, enhancing the textural properties of the oleogels and of the resulting bigels. The hardness of the oleogels ranged from 0.50 N to 1.24 N and for bigels was 5.96-36.75 N. Bigels hardness decreased as the oleogel ratio in the bigel increased. Microscopy and FTIR revealed that the addition of coconut oil in oleogels hampered the formation of a distinct crystalline monoglycerides network. Also, the absence of new peaks in the bigels indicated that the two structured phases interact with each other mostly physically, without the formation of new chemical bonds. Consequently, the oleogels and bigels developed, comprise a promising hard fat substitute with improved nutritional profile.

Structural parameters defining distribution of collagen fiber directions in human carotid arteries.

Collagen fiber arrangement is decisive for constitutive description of anisotropic mechanical response of arterial wall. In this study, their orientation in human common carotid artery was investigated using polarized light microscopy and an automated algorithm giving more than 4·106 fiber angles per slice. In total 113 slices acquired from 18 arteries taken from 14 cadavers were used for fiber orientation in the circumferential-axial plane. All histograms were approximated with unimodal von Mises distribution to evaluate dominant direction of fibers and their concentration parameter. 10 specimens were analyzed also in circumferential-radial and axial-radial planes (2-4 slices per specimen in each plane); the portion of radially oriented fibers was found insignificant. In the circumferential-axial plane, most specimens showed a pronounced unimodal distribution with angle to circumferential direction µ = 0.7° ± 9.4° and concentration parameter b = 3.4 ± 1.9. Suitability of the unimodal fit was confirmed by high values of coefficient of determination (mean R2 = 0.97, median R2 = 0.99). Differences between media and adventitia layers were not found statistically significant. The results are directly applicable as structural parameters in the GOH constitutive model of arterial wall if the postulated two fiber families are unified into one with circumferential orientation.

Independent and combined effects of obesity and traumatic joint injury to the structure and composition of rat knee cartilage.

Osteoarthritis (OA) is a multifactorial joint disease characterized by articular cartilage degradation. Risk factors for OA include joint trauma, obesity, and inflammation, each of which can affect joint health independently, but their interaction and the associated consequences of such interaction were largely unexplored. Here, we studied compositional and structural alterations in knee joint cartilages of Sprague-Dawley rats exposed to two OA risk factors: joint injury and diet-induced obesity. Joint injury was imposed by surgical transection of anterior cruciate ligaments (ACLx), and obesity was induced by a high fat/high sucrose diet. Depth-dependent proteoglycan (PG) content and collagen structural network of cartilage were measured from histological sections collected previously in Collins et al.. (2015). We found that ACLx primarily affected the superficial cartilages. Compositionally, ACLx led to reduced PG content in lean animals, but increased PG content in obese rats. Structurally, ACLx caused disorganization of collagenous network in both lean and obese animals through increased collagen orientation in the superficial tissues and a change in the degree of fibrous alignment. However, the cartilage degradation attributed to joint injury and obesity was not necessarily additive when the two risk factors were present simultaneously, particularly for PG content and collagen orientation in the superficial tissues. Interestingly, sham surgeries caused a through-thickness disorganization of collagen network in lean and obese animals. We conclude that the interactions of multiple OA risk factors are complex and their combined effects cannot be understood by superposition principle. Further research is required to elucidate the interactive mechanism between OA subtypes.

Characteristics of distal femoral articular cartilage in 6 weeks posttraumatic osteoarthritis by a subcritical impact.

Traumatized knee greatly contributes to osteoarthritis (OA) of the knee in young adults. To intervene effectively before the onset of severe structural disruption, detection of the disease at the early onset is crucial. In this study, we put together the findings for the detection of OA from the femoral knee joint cartilage of the rabbit at 6 weeks posttrauma. Articular cartilage samples are taken from the impacted and nonimpacted joints at 0 week (serving as the control group) and at 6 weeks posttrauma by minimal force. The samples were imaged using microscopic magnetic resonance imaging (µMRI) at 11.7 µm/pixel and polarized light microscopy (PLM) at 1 µm/pixel. In addition, an inductively coupled plasma - optical emission spectrometry analysis was performed using the adjacent cartilage samples. The outcomes of this study demonstrate an increase in T2 values in 6 weeks samples compared to the 0 week samples by µMRI technique, indicating a general increase of tissue hydration within cartilage. PLM detects a decrease in the average thickness of the superficial zones in the posttraumatic osteoarthritis samples, significant in the impacted femurs. There was an average increasing trend of maximum retardation in the tide mark in comparison to the reported calcium concentration (mg/L) in impacted samples suggesting a possible rise in mineralization in the 6 weeks samples. Qualitatively, physical observation of the joint after 6 weeks showed signs of reddening in the anterior femur suggesting the disease process is a localized phenomenon. Through microscopic imaging, we are able to detect these changes at 6 weeks posttrauma qualitatively and quantitatively.

Direct measurements of collagen fiber recruitment in the posterior pole of the eye.

Collagen is the main load-bearing component of the peripapillary sclera (PPS) and lamina cribrosa (LC) in the eye. Whilst it has been shown that uncrimping and recruitment of the PPS and LC collagen fibers underlies the macro-scale nonlinear stiffening of both tissues with increased intraocular pressure (IOP), the uncrimping and recruitment as a function of local stretch have not been directly measured. This knowledge is crucial to understanding their functions in bearing loads and maintaining tissue integrity. In this project we measured local stretch-induced collagen fiber bundle uncrimping and recruitment curves of the PPS and LC. Thin coronal samples of PPS and LC of sheep eyes were mounted and stretched biaxially quasi-statically using a custom system. At each step, we imaged the PPS and LC with instant polarized light microscopy and quantified pixel-level (1.5 µm/pixel) collagen fiber orientations. We used digital image correlation to measure the local stretch and quantified collagen crimp by the circular standard deviation of fiber orientations, or waviness. Local stretch-recruitment curves of PPS and LC approximated sigmoid functions. PPS recruited more fibers than the LC at the low levels of stretch. At 10% stretch the curves crossed with 75% bundles recruited. The PPS had higher uncrimping rate and waviness remaining after recruitment than the LC: 0.9º vs. 0.6º and 3.1º vs. 2.7º. Altogether our findings support describing fiber recruitment of both PPS and LC with sigmoid curves, with the PPS recruiting faster and at lower stretch than the LC, consistent with a stiffer tissue. STATEMENT OF SIGNIFICANCE: Peripapillary sclera (PPS) and lamina cribrosa (LC) collagen recruitment behaviors are central to the nonlinear mechanical behavior of the posterior pole of the eye. How PPS and LC collagen fibers recruit under stretch is crucial to develop constitutive models of the tissues but remains unclear. We used image-based stretch testing to characterize PPS and LC collagen fiber bundle recruitment under local stretch. We found that fiber-level stretch-recruitment curves of PPS and LC approximated sigmoid functions. PPS recruited more fibers at a low stretch, but at 10% bundle stretch the two curves crossed with 75% bundles recruited. We also found that PPS and LC fibers had different uncrimping rates and non-zero waviness's when recruited.

Microscopical recognition and characterization of solution dyed fibers.

Solution dyed fibers are synthetic fibers colored through the addition of insoluble pigmentation to the polymer prior to extrusion. This is in contrast to most textile fibers, which are traditionally colored via immersion in liquid dyebaths following extrusion. Solution dyed fibers are increasing in market share in numerous applications (e.g., apparel, carpet, upholstery), and thus, the microscopic characteristics and variety of pigments used to color them represent unexploited properties in forensic fiber comparisons. This paper presents the development of a microscopical process to first recognize a fiber as solution dyed and subsequently characterize the color and optical properties of each type of pigment found in a given fiber. To this end, a set of 76 fibers, representing polypropylene, nylon, polyester, and rayon, spanning all nominal colors and several consumer applications were analyzed longitudinally and in cross section using a combination of polarized light, fluorescence, and oil immersion microscopy. A given fiber contained between one and six different pigments, and a total of 260 pigments (not all unique) were recognized within this set of fibers. Pigment morphologies were categorized as angular, elongated, rounded, finely divided, or streaky, and about 40% of the pigments fluoresced. Ultimately, this body of data is intended to provide trace evidence examiners with a specific approach to recognize and begin to exploit pigmented fibers encountered in casework.

Antimicrobial Efficacy and Dentin Collagen Damage Caused by Calcium Hypochlorite and Sodium Hypochlorite

Abstract This study aimed to evaluate the antimicrobial activity of calcium hypochlorite (Ca (OCl)2) and sodium hypochlorite (NaOCl) using confocal laser scanning microscopy (CLSM) and dentin organic matrix alteration by picrosirius staining and light microscopy (LM). Samples of human extracted teeth were infected with Enterococcus faecalis by centrifugation of the bacterial suspension and were treated with Ca(OCl)2 or NaOCl at 0.5%, 2.5%, and 6% for 15, 30, and 60 seconds. CLSM and viability staining were used to quantitatively analyze the proportions of dead/live bacteria in the canal lumen and border of the root canal. The data were analyzed by ANOVA and Fisher test. For LM analysis, one hundred bovine teeth were randomly divided into 10 test groups (n=10): G1- Without treatment; G2- 17% EDTA; G3- 6% NaOCl; G4- 6% NaOCl + EDTA; G5- 0.5% Ca(OCl)2; G6- 0.5% Ca(OCl)2 + EDTA; G7- 2.5% Ca(OCl)2; G8- 2.5% Ca(OCl)2 + EDTA; G9- 6% Ca(OCl)2; G10- 6% Ca(OCl)2 + EDTA. The samples were fragmented and stained with Picrosirius. Data were analyzed by Kruskal-Wallis and Dunn (P<0.05). There was a strong correlation between the results of the canal lumen and the border of the root canal (r=0.962). Both hypochlorites at a concentration of 0.5% showed less microbial reduction compared to 2.5% and 6% (P<0.05). There was less antimicrobial activity at 15 seconds compared to 30 and 60 seconds (P<0.05). Ca(OCl)2 and NaOCl showed similar results at the same concentrations (P>0.05). In conclusion, Ca(OCl)2 caused fewer alterations to the dentin organic matrix at concentrations of 0.5% and 2.5%. Ca(OCl)2 presents antimicrobial activity similar to NaOCl, and collagen damage is concentration-dependent.

Resumo Este estudo teve como objetivo avaliar a atividade antimicrobiana do hipoclorito de cálcio (Ca(OCl)2) e do hipoclorito de sódio (NaOCl) usando a microscopia confocal a laser (MCVL) e a alteração da matriz orgânica da dentina por meio da coloração com picrosirius e microscopia óptica (MO). Amostras de dentes humanos extraídos foram infectados com Enterococcus faecalis por centrifugação da suspensão bacteriana. As amostras foram tratadas com Ca(OCl)2 ou NaOCl a 0,5%, 2,5% e 6% durante 15, 30 e 60 segundos. A MCVLe a coloração de viabilidade foram utilizadas para analisar quantitativamente as proporções de bactérias vivas/mortas no lúmen do canal e no bordo do canal radicular. Os dados foram analisados por ANOVA e teste de Fisher LSD. Para análise de MO, cem dentes bovinos foram divididos aleatoriamente em 10 grupos teste (n=10): G1- Sem tratamento; G2- 17% de EDTA; G3- 6% de NaOCl; G4- 6% de NaOCl + EDTA; G5- 0,5% de Ca(OCl)2; G6- 0,5% de Ca(OCl)2 + EDTA; G7- 2,5% de Ca(OCl)2; G8- 2,5% de Ca(OCl)2 + EDTA; G9- 6% de Ca(OCl)2; G10- 6% de Ca(OCl)2 + EDTA. As amostras foram fragmentadas e coradas com Picrosirius. Os dados foram analisados por Kruskal-Wallis e teste de Dunn (P<0,05). Houve uma forte correlação nos resultados do lúmen do canal e na borda do canal radicular (r=0,962). Ambos os hipocloritos, a uma concentração de 0,5%, mostraram menor redução microbiana em comparação com 2,5% e 6% (P<0,05). Houve menor atividade antimicrobiana aos 15 segundos em comparação com 30 e 60 segundos (P<0,05). Em conclusão, o Ca(OCl)2 e o NaOCl apresentaram resultados semelhantes nas mesmas concentrações (P>0,05). O Ca(OCl)2 causou menos alterações na matriz orgânica da dentina em concentrações de 0,5% e 2,5%. O Ca(OCl)2 apresenta atividade antimicrobiana semelhante ao NaOCl, e os danos ao colágeno dependem da concentraçã

Broadband scattering properties of articular cartilage zones and their relationship with the heterogenous structure of articular cartilage extracellular matrix.

Significance: Articular cartilage exhibits a zonal architecture, comprising three distinct zones: superficial, middle, and deep. Collagen fibers, being the main solid constituent of articular cartilage, exhibit unique angular and size distribution in articular cartilage zones. There is a gap in knowledge on how the unique properties of collagen fibers across articular cartilage zones affect the scattering properties of the tissue. Aim: This study hypothesizes that the structural properties of articular cartilage zones affect its scattering parameters. We provide scattering coefficient and scattering anisotropy factor of articular cartilage zones in the spectral band of 400 to 1400 nm. We enumerate the differences and similarities of the scattering properties of articular cartilage zones and provide reasoning for these observations. Approach: We utilized collimated transmittance and integrating sphere measurements to estimate the scattering coefficients of bovine articular cartilage zones and bulk tissue. We used the relationship between the scattering coefficients to estimate the scattering anisotropy factor. Polarized light microscopy was applied to estimate the depth-wise angular distribution of collagen fibers in bovine articular cartilage. Results: We report that the Rayleigh scatterers contribution to the scattering coefficients, the intensity of the light scattered by the Rayleigh and Mie scatterers, and the angular distribution of collagen fibers across tissue depth are the key parameters that affect the scattering properties of articular cartilage zones and bulk tissue. Our results indicate that in the short visible region, the superficial and middle zones of articular cartilage affect the scattering properties of the tissue, whereas in the far visible and near-infrared regions, the articular cartilage deep zone determines articular cartilage scattering properties. Conclusion: This study provides scattering properties of articular cartilage zones. Such findings support future research to utilize optical simulation to estimate the penetration depth, depth-origin, and pathlength of light in articular cartilage for optical diagnosis of the tissue.

Comparing age- and bone-related differences in collagen fiber orientation: A case study of bats and laboratory mice using quantitative polarized light microscopy.

As bones age in most mammals, they typically become more fragile. This state of bone fragility is often associated with more homogenous collagen fiber orientations (CFO). Unlike most mammals, bats maintain mechanically competent bone throughout their lifespans, but little is known of positional and age-related changes in CFO within wing bones. This study tests the hypothesis that age-related changes in CFO in big brown bats (Eptesicus fuscus) differ from those of the standard mammalian model for skeletal aging, the C57BL/6 laboratory mouse. We used data from quantitative polarized light microscopy (qPLM) to compare CFO across the lifespan of long-lived big brown bats and age matched C57BL/6 mice. Eptesicus and C57BL/6 mice displayed idiosyncratic patterns of CFO. Consistent age-related changes were only apparent in the outer cortical bone of Eptesicus, where bone tissue is more longitudinally arranged and more anisotropic in older individuals. Both taxa displayed a ring of more transversely oriented bone tissue surrounding the medullary cavity. In Eptesicus, this tissue represents a greater proportion of the overall cross-section, and is more clearly helically aligned (arranged at 45° to the bone long axis) than similar bone tissue in mice. Bat wing bones displayed a proximodistal gradient in CFO anisotropy and longitudinal orientation in both outer and inner cortical bone compartments. This study lays a methodological foundation for the quantitative evaluation of bone tissue architecture in volant and non-volant mammals that may be expanded in the future.

Evaluation of Iontophoresis as a Tool in Comparison to Topical Remineralization Systems by Transverse Microradiography and Polarized Light Microscopy: An In Vitro Study.

Aim: To compare the efficacy of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) (GC Tooth Mousse) and tricalcium phosphate formulation (Clinpro) by topical application and iontophoresis. Background: Noninvasive treatment of dental caries is a major advance in the clinical management of the disease. Materials and methods: A total of 20 primary anterior and 20 primary molars were included in the study to be analyzed by transverse microradiography (TMR) and polarized light microscopy (PLM). The samples were coated with acid-resistant varnish, leaving a window on the buccal/lingual surface of enamel and immersed in demineralizing solution for 96 hours. Each sample was coated with varnish on the left half (control), and the right half served as a test. All the samples were divided into four groups. The test windows of specimens in groups I (GC Tooth Mousse) and II (Clinpro) were subjected to remineralization treatments for 10 days. Similarly, groups III and IV were subjected to iontophoresis using GC Tooth Mousse and Clinpro for 7 minutes. The samples were analyzed by TMR and PLM. Results: There was no significant difference in the mean difference of mineral loss among the four groups. There was a significant difference in the mean difference of lesion depth among the four groups. Conclusion: Both iontophoresis and topical application were equally efficient, but one remineralization by iontophoresis equals 10 topical applications. How to cite this article: VC R, Muppa R, Nallanchakrava S, et al. Evaluation of Iontophoresis as a Tool in Comparison to Topical Remineralization Systems by Transverse Microradiography and Polarized Light Microscopy: An In Vitro Study. Int J Clin Pediatr Dent 2023;16(S-1):S85-S90.

Test characteristics of Raman spectroscopy integrated with polarized light microscopy for the diagnosis of acute gouty arthritis.

OBJECTIVES: We studied the performance of Raman spectroscopy integrated with polarized light microscopy (iRPolM) as a next-generation technique for synovial fluid analysis in gout. METHODS: This is a prospective study, including consecutive synovial fluid samples drawn from any peripheral swollen joint. Diagnostic accuracy was compared to the 2015 ACR/EULAR Gout classification criteria as a reference test and to polarized light microscopy (PLM) analysis by a rheumatologist. Synovial fluid was analysed with iRPolM after unblinding the PLM results. RESULTS: Two hundred unselected consecutive patient samples were included in this study. Validation against clinical criteria: 67 patients were classified as gout according to 2015 ACR/EULAR classification criteria. Compared to the 2015 ACR/EULAR gout classification criteria, iRPolM had a sensitivity of 77.6% (95% CI: 65.8-86.9), specificity of 97.7% (95% CI: 93.5-99.5), positive predictive value (PPV) of 94.5% (95% CI: 84.9-98.2), negative predictive value (NPV) of 89.7% (95% CI: 84.7-93.1), an accuracy of 91.0% (95% CI: 86.2-94.6), a positive likelihood ratio of 34.4 (95% CI: 11.16-106.10) and a negative likelihood ratio of 0.23 (95% CI: 0.15-0.36). Validation against PLM: 55 samples were positive for MSU according to PLM. The interrater agreement between PLM and iRPolM was near perfect (к=0.90). The sensitivity of iRPolM to identify MSU in PLM-positive samples was 91.2% (95% CI: 80.7-97.1), the specificity was 97.6% (95% CI: 93.0-99.5), the PPV was 94.6% (95% CI: 85.0-98.2), NPV was 96.0% (95% CI: 91.2-98.2) and the accuracy was 95.6% (95% CI: 91.4-98.2). The positive likelihood ratio was 37.4 (95% CI: 12.20-114.71), and the negative likelihood ratio was 0.09 (95% CI: 0.04-0.21). CONCLUSION: iRPolM is a promising next-generation diagnostic tool for rheumatology by diagnosing gout with high specificity, increased objectivity, and a sensitivity comparable to PLM.

Calcium oxalate crystal macropattern and its usefulness in the taxonomy of Baccharis (Asteraceae).

This study provides a comprehensive account of the various types of calcium oxalate crystals found in the genus Baccharis and assesses the exceptional value of crystal macropatterns for the taxonomy of the genus. The morphotype, occurrence, and chemical composition of the crystals found in the stems and leaves are studied. The 44 species included in this study were selected based on a broad phylogeny-based sampling covering seven subgenera and 31 sections. These species were chosen to represent all the main phylogenetic lineages of Baccharis; thus, the sampling also represents a comprehensive coverage concerning evolutionary significance for such a large and environmentally and economically important plant group. The samples were analyzed by light microscopy, scanning electron microscopy (SEM), and energy-dispersive x-ray spectroscopy (EDS). Several morphotypes of crystals, including druses, crystal sand, styloids and prisms, were present. Based on their chemical composition, the crystals were classified as pure calcium oxalate, mixtures of oxalates and sulfates, and mixtures of oxalates, sulfates, and silica. The crystal macropatterns observed in this study aid in species identification and provide novel data for the taxonomy of Baccharis. RESEARCH HIGHLIGHTS: Most species of Baccharis have a specific crystalline pattern. Each species produces a crystal morphotype or a set of morphotypes specific to it. The crystals observed are formed by calcium oxalate.

Direct measurements of collagen fiber recruitment in the posterior pole of the eye.

Collagen is the main load-bearing component of the peripapillary sclera (PPS) and lamina cribrosa (LC) in the eye. Whilst it has been shown that uncrimping and recruitment of the PPS and LC collagen fibers underlies the macro-scale nonlinear stiffening of both tissues with increased intraocular pressure (IOP), the uncrimping and recruitment as a function of local stretch have not been directly measured. This knowledge is crucial for the development of constitutive models associating micro and macro scales. In this project we measured local stretch-induced collagen fiber bundle uncrimping and recruitment curves of the PPS and LC. Thin coronal samples of PPS and LC of sheep eyes were mounted and stretched biaxially quasi-statically using a custom system. At each step, we imaged the PPS and LC with instant polarized light microscopy and quantified pixel-level (1.5 µm/pixel) collagen fiber orientations. We used digital image correlation to measure the local stretch and quantified collagen crimp by the circular standard deviation of fiber orientations, or waviness. Local stretch-recruitment curves of PPS and LC approximated sigmoid functions. PPS recruited more fibers than the LC at the low levels of stretch. At 10% stretch the curves crossed with 75% bundles recruited. The PPS had higher uncrimping rate and waviness remaining after recruitment than the LC: 0.9° vs. 0.6° and 3.1° vs. 2.7°. Altogether our findings support describing fiber recruitment of both PPS and LC with sigmoid curves, with the PPS recruiting faster and at lower stretch than the LC, consistent with a stiffer tissue.

Instant polarized light microscopy pi (IPOLπ) for quantitative imaging of collagen architecture and dynamics in ocular tissues.

Collagen architecture determines the biomechanical environment in the eye, and thus characterizing collagen fiber organization and biomechanics is essential to fully understand eye physiology and pathology. We recently introduced instant polarized light microscopy (IPOL) that encodes optically information about fiber orientation and retardance through a color snapshot. Although IPOL allows imaging collagen at the full acquisition speed of the camera, with excellent spatial and angular resolutions, a limitation is that the orientation-encoding color is cyclic every 90 degrees (π/2 radians). In consequence, two orthogonal fibers have the same color and therefore the same orientation when quantified by color-angle mapping. In this study, we demonstrate IPOLπ, a new variation of IPOL, in which the orientation-encoding color is cyclic every 180 degrees (π radians). Herein we present the fundamentals of IPOLπ, including a framework based on a Mueller-matrix formalism to characterize how fiber orientation and retardance determine the color. The improved quantitative capability of IPOLπ enables further study of essential biomechanical properties of collagen in ocular tissues, such as fiber anisotropy and crimp. We present a series of experimental calibrations and quantitative procedures to visualize and quantify ocular collagen orientation and microstructure in the optic nerve head, a region in the back of the eye. There are four important strengths of IPOLπ compared to IPOL. First, IPOLπ can distinguish the orientations of orthogonal collagen fibers via colors, whereas IPOL cannot. Second, IPOLπ requires a lower exposure time than IPOL, thus allowing faster imaging speed. Third, IPOLπ allows visualizing non-birefringent tissues and backgrounds from tissue absorption, whereas both appear dark in IPOL images. Fourth, IPOLπ is cheaper and less sensitive to imperfectly collimated light than IPOL. Altogether, the high spatial, angular, and temporal resolutions of IPOLπ enable a deeper insight into ocular biomechanics and eye physiology and pathology.

Micro-computed Tomography-Based Collagen Orientation and Anisotropy Analysis of Rabbit Articular Cartilage.

The collagen network is the highly organized backbone of articular cartilage providing tissue tensile stiffness and restricting proteoglycan bleaching out of the tissue. Osteoarthritis (OA) diminishes proper collagen network adaptation. Our aim was to provide quantitative three-dimensional (3D) information of the cartilage collagen network adaptation in early osteoarthritis using high resolution micro-computed tomography (µCT)-imaging. Osteochondral samples from the femoral condyles were collected from healthy (N = 8, both legs) and experimental OA rabbit model with anterior cruciate ligament transection (N = 14, single leg). Samples were processed for cartilage µCT-imaging and histological evaluation with polarized light microscopy (PLM). Structure tensor analysis was used to analyse the collagen fibre orientation and anisotropy of the µCT-images, and PLM was used as a validation for structural changes. Depth-wise comparison of collagen fibre orientation acquired with µCT-imaging and PLM correlated well, but the values obtained with PLM were systematically greater than those measured with µCT-imaging. Structure tensor analysis allowed for 3D quantification of collagen network anisotropy. Finally, µCT-imaging revealed only minor differences between the control and experimental groups.

T2orientation anisotropy mapping of articular cartilage using qMRI.

Objective.To provide orientation-independent MR parameters potentially sensitive to articular cartilage degeneration by measuring isotropic and anisotropic components ofT2relaxation, as well as 3D fiber orientation angle and anisotropy via multi-orientation MR scans.Approach. Seven bovine osteochondral plugs were scanned with a high angular resolution of thirty-seven orientations spanning 180° at 9.4 T. The obtained data was fitted to the magic angle model of anisotropicT2relaxation to produce pixel-wise maps of the parameters of interest. Quantitative Polarized Light Microscopy (qPLM) was used as a reference method for the anisotropy and fiber orientation.Main results. The number of scanned orientations was found to be sufficient for estimating both fiber orientation and anisotropy maps. The relaxation anisotropy maps demonstrated a high correspondence with qPLM reference measurements of the collagen anisotropy of the samples. The scans also enabled calculating orientation-independentT2maps. Little spatial variation was observed in the isotropic component ofT2while the anisotropic component was much faster in the deep radial zone of cartilage. The estimated fiber orientation spanned the expected 0°-90° in samples that had a sufficiently thick superficial layer. The orientation-independent magnetic resonance imaging (MRI) measures can potentially reflect the true properties of articular cartilage more precisely and robustly.Significance. The methods presented in this study will likely improve the specificity of cartilage qMRI by allowing the assessment of the physical properties such as orientation and anisotropy of collagen fibers in articular cartilage.

Dependence of crack shape in loaded articular cartilage on the collagenous structure.

Cartilage cracks disrupt tissue mechanics, alter cell mechanobiology, and often trigger tissue degeneration. Yet, some tissue cracks heal spontaneously. A primary factor determining the fate of tissue cracks is the compression-induced mechanics, specifically whether a crack opens or closes when loaded. Crack deformation is thought to be affected by tissue structure, which can be probed by quantitative polarized light microscopy (PLM). It is unclear how the PLM measures are related to deformed crack morphology. Here, we investigated the relationship between PLM-derived cartilage structure and mechanical behavior of tissue cracks by testing if PLM-derived structural measures correlated with crack morphology in mechanically indented cartilages. METHODS: Knee joint cartilages harvested from mature and immature animals were used for their distinct collagenous fibrous structure and composition. The cartilages were cut through thickness, indented over the cracked region, and processed histologically. Sample-specific birefringence was quantified as two-dimensional (2D) maps of azimuth and retardance, two measures related to local orientation and degree of alignment of the collagen fibers, respectively. The shape of mechanically indented tissue cracks, measured as depth-dependent crack opening, were compared with azimuth, retardance, or "PLM index," a new parameter derived by combining azimuth and retardance. RESULTS: Of the three parameters, only the PLM index consistently correlated with the crack shape in immature and mature tissues. CONCLUSION: In conclusion, we identified the relative roles of azimuth and retardance on the deformation of tissue cracks, with azimuth playing the dominant role. The applicability of the PLM index should be tested in future studies using naturally-occurring tissue cracks.