Naturally Derived Luminescent Material in Engineered Silk and Its Application as a Fluorescent Dye with a Large Stokes Shift and Sensing Capability.

Silkworms have provided valuable byproducts (spanning from high-quality textiles to health supplements) to humans for millennia. Despite their importance in sericultural economy and biotechnology, manifold possibilities inherent in the myriad natural or artificially generated silk varieties have been underestimated. In this paper, we report that the Yeonnokjam silk strain, which shows light-green color, contains quercetin fluorochrome (QueF) in sericin, and QueF can be used as a fluorescence dye with a large Stokes shift and high sensitivity to environmental temperature and pH, thus functioning as an environmental sensing material. A Stokes shift exceeding 180 nm, a quantum efficiency of 1.28%, and a rapid fluorescence decay of 0.67 ns are obtained, which are influenced by solvent polarities. Moreover, QueF can be used as a UV blocker as well, and its low cytotoxicity and biocompatibility further suggest promising prospects for diverse application in cosmetics and medical materials in the future.

The fluorochrome-to-protein ratio is crucial for the flow cytometric detection of tissue factor on extracellular vesicles.

Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.

Visualization of bone formation in sheep's middle ear by using fluorochrome sequential labelling (FSL).

One factor for the lacking integration of the middle ear stapes footplate prosthesis or the missing healing of stapes footplate fractures could be the known osteogenic inactivity. In contrast, it was recently demonstrated that titanium prostheses with an applied collagen matrix and immobilised growth factors stimulate osteoblastic activation and differentiation on the stapes footplate. Regarding those findings, the aim of this study was to evaluate the potential of bone regeneration including bone remodeling in the middle ear. Ten one-year-old female merino sheep underwent a middle ear surgery without implantation of middle ear prostheses or any other component for activating bone formation. Post-operatively, four fluorochromes (tetracycline, alizarin complexion, calcein green and xylenol orange) were administered by subcutaneous injection at different time points after surgery (1 day: tetracycline, 7 days: alizarin, 14 days: calcein, 28 days: xylenol). After 12 weeks, the temporal bones including the lateral skull base were extracted and histologically analyzed. Fluorescence microscopy analysis of the entire stapes with the oval niche, but in particular stapes footplate and the Crura stapedis revealed evidence of new bone formation. Calcein was detected in all and xylenol in 60% of the animals. In contrast, tetracycline and alizarin could only be verified in two animals. The authors were able to demonstrate the osseoregenerative potential of the middle ear, in particular of the stapes footplate, using fluorescence sequence labelling.

Osseointegration of photodynamic active biomaterials for bone regeneration in an animal bone model over a period of 12 months.

OBJECTIVES: Previous efforts led to the development of two different polymeric biomaterials for periodontal regeneration with antibacterial photodynamic surface activity. The present study aimed to investigate osseointegration and bone formation of both materials in an ovine model. METHODS: Both biomaterials: 1) urethane dimethacrylate-based Biomaterial 1 (BioM1) and 2) tri-armed oligoester-urethane methacrylate-based Biomaterial 2 (BioM2) are enriched with beta-tri-calcium phosphate and the photosensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC). These materials were implanted in non-critical size bone defects in the sheep femur (n = 16) and tibia (n = 8). Empty defects served as controls (n = 16). Polyfluorochrome sequential bone labeling was carried out at baseline and after 3, 6, and 12 months. Animals were sacrificed after 12 months. Bone specimens (n = 40) were fixed and subjected to microtomographic analysis (µCT) for the evaluation of the bone-volume-fraction (BV/TV), trabecular number and trabecular thickness. Subsequently, histological sections were arranged and polyfluorochrome sequential bone labeling was analyzed by confocal laser scanning microscopy (cLSM). RESULTS: cLSM analysis revealed that highest remodeling and bone formation activity occurred during the second half of the study period (6-12 months). Bone formation in the tibia was significantly lower for the control (2.71 ± 1.26%) as compared to BioM1 (6.01 ± 2.99%) and BioM2 (6.45 ± 2.12%); (p = 0.006, p = 0004). Micro-computed tomography revealed a BV/TV volume fraction of 44.72 ± 9.01% in femur defects filled with BioM1 which was significantly higher compared to the control (32.27 ± 7.02%; p = 0.01). Bone architecture (trabecular number, trabecular thickness) did not significantly differ from the self-healed defects. SIGNIFICANCE: Both biomaterials, especially BioM1 showed good osseointegration and bone formation characteristics and can be recommended for further examination in periodontal regeneration studies.

Comparative Cytogenetics and Fluorescent Chromosome Banding in Five Indian Species of Dipcadi Medik.

The genus Dipcadi Medik. (Subfamily: Scilloideae) has a narrow distribution in India and several overlapping morphological traits make the genus taxonomically challenging at the species level. Cytogenetic characterization can provide additional taxonomic data and can be used to evaluate genetic diversity at the species level. We have accomplished comparative karyotype analysis and fluorescence banding patterns using 4'-6-Diamidino-2-phenylindole (DAPI) and Chromomycin A3 (CMA) in five Indian species for the first time. The karyotypes of D. concanense and D. goaense exhibited similar fluorochrome banding profiles. However, D. montanum, D. ursulae and D. erythraeum differ distinctly in their karyotypes. In all taxa, CMA+ve/DAPI-ve or DAPI0 (GC-rich) constitutive heterochromatin was located at the constriction region or terminal satellite of the nucleolar chromosome. DAPI+ve/CMA-ve or CMA0 (AT-rich) heterochromatin dominates in D. montanum, D. ursulae and D. erythraeum. However, D. erythraeum shows a distinct variation in fluorochrome banding pattern from all other species. The distribution of CMA and DAPI bands is a reflection of heterochromatin composition and variations acquired by different species. This characterization can be used to assess phylogenetic relationships in the understudied genus Dipcadi and may serve as a basis for other genomic analyses and evolutionary studies.

Comparative karyotype analysis of eight Cucurbitaceae crops using fluorochrome banding and 45S rDNA-FISH.

To have an insight into the karyotype variation of eight Cucurbitaceae crops including Cucumissativus Linnaeus, 1753, Cucumismelo Linnaeus, 1753, Citrulluslanatus (Thunberg, 1794) Matsumura et Nakai, 1916, Benincasahispida (Thunberg, 1784) Cogniaux, 1881, Momordicacharantia Linnaeus, 1753, Luffacylindrica (Linnaeus, 1753) Roemer, 1846, Lagenariasicerariavar.hispida (Thunberg, 1783) Hara, 1948 and Cucurbitamoschata Duchesne ex Poiret, 1819, well morphologically differentiated mitotic metaphase chromosomes were prepared using the enzymatic maceration and flame-drying method, and the chromosomal distribution of heterochromatin and 18S-5.8S-26S rRNA genes (45S rDNA) was investigated using sequential combined PI and DAPI (CPD) staining and fluorescence in situ hybridization (FISH) with 45S rDNA probe. Detailed karyotypes were established using the dataset of chromosome measurements, fluorochrome bands and rDNA FISH signals. Four karyotype asymmetry indices, CVCI, CVCL, MCA and Stebbins' category, were measured to elucidate the karyological relationships among species. All the species studied had symmetrical karyotypes composed of metacentric and submetacentric or only metacentric chromosomes, but their karyotype structure can be discriminated by the scatter plot of MCA vs. CVCL. The karyological relationships among these species revealed by PCoA based on x, 2n, TCL, MCA, CVCL and CVCI was basically in agreement with the phylogenetic relationships revealed by DNA sequences. CPD staining revealed all 45S rDNA sites in all species, (peri)centromeric GC-rich heterochromatin in C.sativus, C.melo, C.lanatus, M.charantia and L.cylindrica, terminal GC-rich heterochromatin in C.sativus. DAPI counterstaining after FISH revealed pericentromeric DAPI+ heterochromatin in C.moschata. rDNA FISH detected two 45S loci in five species and five 45S loci in three species. Among these 45S loci, most were located at the terminals of chromosome arms, and a few in the proximal regions. In C.sativus, individual chromosomes can be precisely distinguished by the CPD band and 45S rDNA signal patterns, providing an easy method for chromosome identification of cucumber. The genome differentiation among these species was discussed in terms of genome size, heterochromatin, 45S rDNA site, and karyotype asymmetry based on the data of this study and previous reports.

Wild boar versus domestic pig-Deciphering of crown growth in porcine second molars.

Based on the previously established periodicity of enamel growth marks, we reconstructed crown growth parameters of mandibular second molars from two wild boar and two domestic pigs of the Linderöd breed. Body weight gain and progression of dental development were markedly faster in the domestic pigs than the wild boar. While the final crown dimensions of the M2 did not differ between domestic pigs and wild boar, mean crown formation time (CFT) of this tooth was considerably shorter in the domestic pigs (162 days) than in the wild boar (205 days). The difference in CFT was mainly attributable to a higher enamel extension rate (EER) in the domestic pig. Generally, EER was highest in the cuspalmost deciles of the length of the enamel-dentine-junction and markedly dropped in cervical direction, with lowest values occurring in the cervicalmost decile. In consequence, the cuspal half of the M2 crown was formed about three times faster than the cervical half. In contrast to the EER, no marked difference in daily enamel secretion rate (DSR) was recorded between domestic pigs and wild boar. The duration of enamel matrix apposition as well as linear enamel thickness in corresponding crown portions was only slightly lower in the domestic pigs than the wild boar. Thus, the earlier completion of M2 crown growth in the domestic pig was mainly achieved by a higher EER and not by an increased DSR. The more rapid recruitment of secretory ameloblasts in the course of molar crown formation of domestic pigs compared to wild boar is considered a side-effect of the selection for rapid body growth during pig domestication.

Concurrent bioimaging of microalgal photophysiology and oxidative stress.

The production of reactive oxygen species (ROS) is an unavoidable consequence of oxygenic photosynthesis and represents a major cause of oxidative stress in phototrophs, having detrimental effects on the photosynthetic apparatus, limiting cell growth, and productivity. Several methods have been developed for the quantification of cellular ROS, however, most are invasive, requiring the destruction of the sample. Here, we present a new methodology that allows the concurrent quantification of ROS and photosynthetic activity, using the fluorochrome dichlorofluorescein (DCF) and in vivo chlorophyll a fluorescence, respectively. Both types of fluorescence were measured using an imaging Pulse Amplitude Modulation (PAM) fluorometer, modified by adding a UVA-excitation light source (385 nm) and a green bandpass emission filter (530 nm) to enable the sequential capture of red chlorophyll fluorescence and green DCF fluorescence in the same sample. The method was established on Phaeodactylum tricornutum Bohlin, an important marine model diatom species, by determining protocol conditions that permitted the detection of ROS without impacting photosynthetic activity. The utility of the method was validated by quantifying the effects of two herbicides (DCMU and methyl viologen) on the photosynthetic activity and ROS production in P. tricornutum and of light acclimation state in Navicula cf. recens Lange-Bertalot, a common benthic diatom. The developed method is rapid and non-destructive, allowing for the high-throughput screening of multiple samples over time.

Sodium Multivitamin Transporter-Targeted Fluorochrome Facilitates Enhanced Metabolic Evaluation of Tumors Through Coenzyme-R Dependent Intracellular Signaling Pathways.

BACKGROUND: Intraoperative molecular imaging (IMI)-guided resections have been shown to improve oncologic outcomes for patients undergoing surgery for solid malignancies. The technology utilizes fluorescent tracers targeting cancer cells without the use of any ionizing radiation. However, currently available targeted IMI tracers are effective only for tumors with a highly specific receptor expression profile, and there is an unmet need for IMI tracers to label a broader range of tumor types. Here, we describe the development and testing of a novel tracer (CR)-S0456) targeted to the sodium multivitamin transporter (SMVT). METHODS: Preclinical models of fibrosarcoma (HT-1080), lung (A549), breast (4T1), and renal cancers (HEK-293 T) in vitro and in vivo were used for assessment of (CR)-S0456 specific tumor labeling via sodium-mediated SMVT uptake in dipotassium phosphate or choline chloride-containing media buffer. Additionally, pharmacologic inhibition of multiple intracellular coenzyme-R obligate signaling pathways, including holocarboxylase synthetase (sulconazole nitrate), PI3K/AKT/mTOR (omipalisib), and calmodulin-dependent phosphatase (calmidazolium), were investigated to assess (CR)-S0456 uptake kinetics. Human fibrosarcoma-bearing xenografts in athymic nude mice were used for tumor and metabolic-specific labeling. Novel NIR needle confocal laser endomicroscopic (nCLE) intratumoral sampling was performed to demonstrate single-cell specific labeling by CR-S0456. RESULTS: CR-S0456 localization in vitro correlated with highly proliferative cell lines (MTT) and doubling time (p < 0.05) with the highest microscopic fluorescence detected in aggressive human fibrosarcomas (HT-1080). Coenzyme-R-specific localization was demonstrated to be SMVT-specific after competitive inhibition of internal localization with excess administration of pantothenic acid. Inhibiting the activity of SMVT by affecting sodium ion hemostasis prevented the complete uptake of CR-S0456. In vivo validation demonstrated (CR)-S0456 localization to xenograft models with accurate identification of primary tumors as well as margin assessment down to 1 mm3 tumor volume. Systemic treatment of xenograft-bearing mice with a dual PI3K/mTOR inhibitor suppressed intratumoral cell signaling and (CR)-S0456 uptake via a reduction in SMVT expression. Novel analysis of in vivo intratumoral cytologic fluorescence using near-infrared confocal laser endomicroscopy demonstrated the absence of coenzyme-R-mediated NIR fluorescence but not fibroblast activation protein (FAP)-conjugated fluorochrome, indicating specific intracellular inhibition of coenzyme-R obligate pathways. CONCLUSION: These findings suggest that a SMVT-targeted NIR contrast agent can be a suitable tracer for imaging a wide range of malignancies as well as evaluating metabolic response to systemic therapies, similar to PET imaging with immune checkpoint inhibitors.

Staining Methods for Normal and Regenerative Myelin in the Nervous System.

Histochemical and fluorescence-based techniques enable the specific identification of myelin by bright-field or fluorescence microscopy. In this chapter, we describe four histological methods for the evaluation of myelin on peripheral nerve tissue sections. The first method combines the Luxol fast blue (LFB) technique with a modified Picrosirius staining contrasted with Harris hematoxylin, called MCOLL. This method simultaneously stains myelin, collagen fibers, and cell nuclei, thus giving an integrated overview of the histology, collagen network, and myelin content of the tissue in paraffin-embedded or cryosectioned samples. Secondly, we describe the osmium tetroxide method, which provides a permanent positive reaction for myelin as well as other lipids present in the tissue. The third method is the immunofluorescence-based detection of myelin proteins that allows to combine information about their expression status with other proteins of interest. Finally, the FluoroMyelin™ stains enable a fast detection of the myelin content that can be easily implemented in immunofluorescence staining panels for cryosectioned tissues. Together, this chapter provides a variety of methods to accurately identify myelin in different experimental approaches.

Genetic Tools for Analyzing Foxp3+ Treg Cells: Fluorochrome-Based Transcriptional Reporters and Genetic Fate-Mapping.

The lack of unambiguous Foxp3+ Treg cell-specific surface markers has prompted the development of various transgenic mouse lines with Foxp3-dependent reporter activity, which involved different fluorochromes and transgenic strategies, including coexpression of multiple transgenes, such as Cre recombinase. Since then, Foxp3 transcriptional reporter has proven to be an indispensable tool to identify and isolate viable Foxp3+ Treg cell populations. However, the physiologic Treg cell pool is functionally heterogeneous and consists of intrathymically (tTreg) and peripherally (pTreg) induced Treg cells, which may confound interpretation of data relying on indiscriminatory Foxp3-fluorochrome reporter expressed in all Treg cells. In this chapter, we describe how the dual Foxp3RFP/GFP reporter can be exploited to discriminate both developmental sublineages based on tTreg cell lineage-specific GFP/Cre recombinase activity, in conjunction with Foxp3-driven RFP expression in all Foxp3+ Treg cells, and provide guidelines for experimental design and implementation. We also elaborate on the possibility to exploit GFP/Cre expression of Foxp3RFP/GFP reporter mice for the manipulation of gene expression (activation and inactivation), such as lineage tracing and in vivo ablation of tTreg cells, while sparing pTreg cells.

Precision Surgery Guided by Intraoperative Molecular Imaging.

Intraoperative molecular imaging (IMI) has recently emerged as an important tool in the armamentarium of surgical oncologists. IMI allows real-time assessment of oncologic resection quality, margin assessment, and occult disease detection during real-time surgery. Numerous tracers have now been developed for use in IMI-guided tissue sampling. Fluorochromes localize to the tumor by taking advantage of their disorganized capillary milieu, overexpressed receptors, or upregulated enzymes. Although fluorescent tracers can suffer from issues of autofluorescence and lack of depth penetration, these challenges are being addressed through hybrid radioactive/fluorescent tracers and new tracers that fluoresce in the near-infrared (NIR-II [wavelength > 1,000 nm]) range. IMI is already being used to treat numerous cancers, with demonstrated improvement in cancer recurrence and patient outcomes without incurring significant burden on either clinicians or patients. In this comprehensive review, we discuss history, mechanism, current oncologic applications, and future directions of IMI-guided optical biopsy.

A Multiplex PCR Approach to Determine Vegetative Incompatibility Genotypes and Mating Type in Cryphonectria parasitica.

This chapter presents a genotyping assay that uses DNA isolated from axenic cultures of Cryphonectria parasitica, which discriminates the six known diallelic vic loci and the two mating idiomorphs (MAT gene) based on (i) modified primer, labeled with a fluorescent dye, (ii) multiplex polymerase chain reaction (multiplex-PCR), and (iii) capillary electrophoresis system. Alternatively, we show that the same primer set is suitable for conventional PCR of each vic locus and MAT gene using nonmodified primer and agarose gel electrophoresis.

Unlocking autofluorescence in the era of full spectrum analysis: Implications for immunophenotype discovery projects.

Understanding the complex elements affecting signal resolution in cytometry is key for quality experimental design and data. In this study, we incorporate autofluorescence as a contributing factor to our understanding of resolution in cytometry and corroborate its impact in fluorescence signal detection through mathematical predictions supported by empirical evidence. Our findings illustrate the critical importance of autofluorescence extraction via full spectrum unmixing in unmasking dim signals and delineating the expression and subset distribution of low abundance markers in discovery projects. We apply our findings to the precise definition of the tissue and cellular distribution of a weakly expressed fluorescent protein that reports on a low-abundance immunological gene. Exploiting the full spectrum coverage enabled by Aurora 5L, we describe a novel approach to the isolation of pure cell subset-specific autofluorescence profiles based on high dimensionality reduction algorithms. This method can also be used to unveil differences in the autofluorescent fingerprints of tissues in homeostasis and after immunological challenges.

Novel PE and APC tandems: Additional near-infrared fluorochromes for use in spectral flow cytometry.

Recent advances in flow cytometry instrumentation and fluorochrome chemistries have greatly increased fluorescent conjugated antibody combinations that can be used reliably and easily in routine experiments. The Cytek Aurora flow cytometer was first released with three excitation lasers (405, 488, and 640 nm) and incorporated the latest Avalanche Photodiode (APD) technology, demonstrating significant improvement in sensitivity for fluorescent emission signals longer than 800 nm. However, there are limited commercially available fluorochromes capable of excitation with peak emission signals beyond 800 nm. To address this gap, we engineered six new fluorochromes: PE-750, PE-800, PE-830 for the 488 nm laser and APC-750, APC-800, APC-830 for the 640 nm laser. Utilizing the principal of fluorescence resonance energy transfer (FRET), these novel structures were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Additionally, each of these fluorochrome conjugates were shown to be compatible with fixation/permeabilization buffer reagents, and demonstrated acceptable brightness and stability when conjugated to antigen-specific monoclonal antibodies. These six novel fluorochrome reagents can increase the numbers of fluorochromes that can be used on a spectral flow cytometer.

Adsorption of PCE in alkali-activated materials analysed by fluorescence microscopy.

The realisation of high-performance concrete mixtures requires the use of superplasticizers to achieve a low water/binder ratio and thus high strengths. Polycarboxylate ethers (PCE) are mostly used as superplasticizers. The effectiveness of these superplasticizers depends on their chemical structure, the binders' alkaline environment and the ions present in the pore solution of the binder. In high alkaline systems like some alkali-activated materials no effective superplasticizer have been found yet. To unravel the compatibility of certain PCE to such a highly alkaline system a fluorescence microscopy approach was used. In first experiments, the adsorption of APEG (allyl ether) and MPEG (methacrylate) PCE on ground granulated blast furnace slag and fly ash was investigated varying the concentration of the activators. At a certain concentration, a complexation of the PCE can be recognised in fluorescence microscope. APEG shows a better stability compared to MPEG; this correlates with rheological investigations.

Functional Adaptation of LPS-affected Dentoalveolar Fibrous Joints in Rats.

INTRODUCTION: The functional interplay between cementum of the root and alveolar bone of the socket is tuned by a uniquely positioned 70-80 µm wide fibrous and lubricious ligament in a dentoalveolar joint (DAJ). In this study, structural and biomechanical properties of the DAJ, periodontal ligament space (PDL-space also known as the joint space), alveolar bone of the socket, and cementum of the tooth root that govern the biomechanics of a lipopolysaccharide (LPS)-affected DAJ were mapped both in space and time. METHODS: The hemi-maxillae from 20 rats (4 control at 6 weeks of age, 4 control and 4 LPS-affected at 12 weeks of age, 4 control and 4 LPS-affected at 16 weeks of age) were investigated using a hybrid technique; micro-X-ray computed tomography (5 µm resolution) in combination with biomechanical testing in situ. Temporal variations in bone and cementum volume fractions were evaluated. Trends in mineral apposition rates (MAR) in additional six Sprague Dawley rats (3 controls, 3 LPS-affected) were revealed by transforming spatial fluorochrome signals to functional growth rates (linearity factor - RW) of bone, dentin, and cementum using a fast Fourier transform on fluorochrome signals from 100-µm hemi-maxillae sections. RESULTS: An overall change in LPS-affected DAJ biomechanics (a 2.5-4.5X increase in tooth displacement and 2X tooth rotation at 6 weeks, no increase in displacement and a 7X increase in rotation at 12 weeks; 27% increase in bone effective strain at 6 weeks and 11% at 12 weeks relative to control) was associated with structural changes in the coronal regions of the DAJ (15% increase in PDL-space from 0 to 6 weeks but only 5% from 6 to 12 weeks compared to control). A significant increase (p < 0.05) in PDL-space between ligated and age-matched control was observed. The bone fraction of ligated at 12 weeks was significantly lower than its age-matched control, and no significant differences (p > 0.05) between groups were observed at 6 weeks. Cementum in the apical regions grew faster but nonlinearly (11% and 20% increase in cementum fraction (CF) at 6 and 12 weeks) compared to control. Alveolar bone revealed site-specific nonlinear growth with an overall increase in MAR (108.5 µm/week to 126.7 µm/week after LPS treatment) compared to dentin (28.3 µm/week in control vs. 26.1 µm/week in LPS-affected) and cementum (126.5 µm/week in control vs. 119.9 µm/week in LPS-affected). A significant increase in CF (p < 0.05) in ligated specimens was observed at 6 weeks of age. CONCLUSIONS: Anatomy-specific responses of cementum and bone to the mechano-chemo stimuli, and their collective temporal contribution to observed changes in PDL-space were perpetuated by altered tooth movement. Data highlight the "resilience" of DAJ function through the predominance of nonlinear growth response of cementum, changes in PDL-space, and bone architecture. Despite the significant differences in bone and cementum architectures, data provided insights into the reactionary effects of cementum as a built-in compensatory mechanism to reestablish functional competence of the DAJ. The spatial shifts in architectures of alveolar bone and cementum, and consequently ligament space, highlight adaptations farther away from the site of insult, which also is another novel insight from this study. These adaptations when correlated within the context of joint function (biomechanics) illustrate that they are indeed necessary to sustain DAJ function albeit being pathological.

Quadruple node of triple junctions of grain boundaries in a Eu2+-doped solid solution of the ions K+, Rb+, Cl- and Br-: an epifluorescence microscopy study using the doping ion as a fluorochrome.

Epifluorescence microscopy was used to unveil the geometry and relative spatial orientation of an arrangement of crystal defects, consisting of a quadruple node of triple junctions of grain boundaries, in a Eu2+-doped solid solution of K+, Rb+, Cl- and Br-. The doping ion was utilized as a fluorochrome. Microscopy images of different optical cross-sections of the arrangement of crystal defects under study were recorded and used to build an electronic three-dimensional reconstruction of this arrangement. The geometry is that of an irregular tristetrahedron, centred at the quadruple node, so that the tristetrahedron legs, deviating from the lattice <111>-zone axis directions, lie along the triple junctions, whereas the tristetrahedron faces collapse onto the grain boundaries. The deviation angles as well as the angles defined by different triple junction pairs and different grain boundary pairs were measured. The orientational deviation is larger than that in a KI single crystal but lower than that in a solid solution of K+, Cl- and Br-, meaning that the structural equilibrium depends on the matrix lattice structural character. Uncompensated local nanostrains, due to the ion substitution, are associated with the observed decrement in structural stability, in relation to the KI case, whereas compensated local nanostrains, provoked by the simultaneous substitution of cations and anions, are argued to be responsible for the observed increment in structural stability, in relation with the K(Cl, Br) case. The optical characterization of the fluorochrome, the recording and processing of the microscopy images, the building of the electronic 3D reconstruction, the angular measure methodology and the geometrical modelling are all carefully described.

Seasonal Growth of the Purple Sea Urchin Heliocidaris crassispina Revealed by Sequential Fluorochrome Tagging.

Many studies have applied fluorochrome tagging to examine the growth of animals with calcified skeletons, but most of them have used only a single tag to determine the annual growth rate. We used sequential fluorochrome tagging to study the seasonal growth of the purple sea urchin Heliocidaris crassispina in Hong Kong waters from February 2012 to February 2013. Sea urchins ranging from 18.9 to 42.7 mm in test diameter had a yearly growth from 0.6 to 13.0 mm. During that year, the sea urchins grew from 0.6 to 5.0 mm in test diameter during the first six months, and from 0.4 to 10.2 mm in test diameter in the second six months. The seasonal differences in growth were confirmed using the von Bertalanffy model. The growth was clear for young sea urchins, especially for individuals less than 5 years old, but was not evident for sea urchins older than 7 years. The seasonal differences in growth were probably related to the reproductive cycle and the seasonal differences in environmental conditions. Our empirical results provide the first evidence of seasonal changes in growth for H. crassispina, demonstrating the usefulness of sequential fluorochrome tagging in studying the growth of sea urchins in the field. We also identify the problem of low recovery of tagged individuals and provide recommendations to improve the tagging procedure.

Six-Color Confocal Immunofluorescence Microscopy with 4-Laser Lines.

Confocal immunofluorescence microscopy is an advanced imaging technique routinely applied in the laboratory and clinics. Histological analyses are performed from tissue material. In general, a single fluorochrome per laser is employed, limiting simultaneous analysis to four antigens in one staining with a conventional 4-laser line microscope. Here, we describe a protocol for combining fluorochromes with the same excitation but different emission properties that allows for the analysis of six different antigens in confocal immunofluorescence microscopy with a conventional 4-laser line microscope. The proposed multiplexed method permits the identification and characterization of complex cell populations in rare tissue material.