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Boron dipyrromethene dyes are highly attractive for image-guided photodynamic therapy. Nevertheless, their clinical breakthrough as theranostic agents is still obstructed by several limitations. Here, we report a series of strongly absorbing, heavy-atom-free, distyryl-BODIPY donor-acceptor dyads operating within the phototherapeutic window. Whereas diphenylamine and carbazole donors lead to strong fluorescence, dimethylacridine, phenoxazine, and phenothiazine units afford a decent fluorescence combined with the efficient formation of singlet oxygen. Dedicated photophysical analysis and quantum-chemical calculations are performed to elucidate the excited state dynamics responsible for the pronounced differences within the BODIPY series. Femtosecond transient absorption spectra reveal the nature of the excited state processes and the involvement of charge-transfer states in triplet formation. The authors thank Hasselt University and the University of Namur for continuing financial support (PhD scholarships JD and TC). BC and WM thank the Research Foundation -Flanders (FWO) for support through projects G087718N, G0D1521N, I006320N, GOH3816NAUHL, and the Scientific Research Community 'Supramolecular Chemistry and Materials' (W000620N). The calculations were performed on the computers of the 'Consortium des equipements de Calcul Intensif (CE ' CI)' (https://www.ceci-hpc.be), including those of the 'UNamur Technological Platform of High-Performance Computing (PTCI)' (https://www.ptci.unamur.be), for which we gratefully acknowledge financial support from the FNRS-FRFC, the Walloon Region, and the University of Namur (Conventions no. 2.5020.11, GEQ U.G006.15, U.G018.19, 1610468, and RW/GEQ2016). SD, AL, and MDD acknowledge support from the European Union's Horizon 2020 research and innovation program under grant agreement no. 871124 Laserlab-Europe.

BACKGROUND: The gut microbiome plays an essential role in human health. Despite the link between air pollution exposure and various diseases, its association with the gut microbiome during susceptible life periods remains scarce. OBJECTIVES: In this study, we examined the association between black carbon particles quantified in prenatal and postnatal biological matrices and bacterial richness and diversity measures, and bacterial families. METHODS: A total of 85 stool samples were collected from 4- to 6-y-old children enrolled in the ENVIRonmental influence ON early AGEing birth cohort. We performed 16S rRNA gene sequencing to calculate bacterial richness and diversity indices (Chao1 richness, Shannon diversity, and Simpson diversity) and the relative abundance of bacterial families. Black carbon particles were quantified via white light generation under femtosecond pulsed laser illumination in placental tissue and cord blood, employed as prenatal exposure biomarkers, and in urine, used as a post-natal exposure biomarker. We used robust multivariable-adjusted linear models to examine the associations between quantified black carbon loads and measures of richness (Chao1 index) and diversity (Shannon and Simpson indices), adjusting for parity, season of delivery, sequencing batch, age, sex, weight and height of the child, and maternal education. Additionally, we performed a differential relative abundance analysis of bacterial families with a correction for sampling fraction bias. Results are expressed as percentage difference for a doubling in black carbon loads with 95% confidence interval (CI). RESULTS: Two diversity indices were negatively associated with placental black carbon [Shannon: -4.38% (95% CI: -8.31%, -0.28%); Simpson: -0.90% (95% CI: -1.76%, -0.04%)], cord blood black carbon [Shannon: -3.38% (95% CI: -5.66%, -0.84%); Simpson: -0.91 (95% CI: -1.66%, -0.16%)], and urinary black carbon [Shannon: -3.39% (95% CI: -5.77%, -0.94%); Simpson: -0.89% (95% CI: -1.37%, -0.40%)]. The explained variance of black carbon on the above indices varied from 6.1% to 16.6%. No statistically significant associations were found between black carbon load and the Chao1 richness index. After multiple testing correction, placental black carbon was negatively associated with relative abundance of the bacterial families Defluviitaleaceae and Marinifilaceae, and urinary black carbon with Christensenellaceae and Coriobacteriaceae; associations with cord blood black carbon were not statistically significant after correction. CONCLUSION: Black carbon particles quantified in prenatal and postnatal biological matrices were associated with the composition and diversity of the childhood intestinal microbiome. These findings address the influential role of exposure to air pollution during pregnancy and early life in human health. https://doi.org/10.1289/EHP11257. ispartof: Environ Health Perspect vol:131 issue:1 pages:17010- ispartof: location:United States status: published

BackgroundPregnant women and developing fetuses comprise a particularly vulnerable population as multiple studies have shown associations between prenatal air pollution exposure and adverse pregnancy outcomes. However, the mechanisms underlying the observed developmental toxicity are mostly unknown, in particular, if pollution particles can cross the human placenta to reach the fetal circulation.ResultsHere, we investigated the accumulation and translocation of diesel exhaust particles (DEPs), as a model particle for combustion-derived pollution, in human perfused placentae using label-free detection by femtosecond pulsed laser illumination. The results do not reveal a significant particle transfer across term placentae within 6 h of perfusion. However, DEPs accumulate in placental tissue, especially in the syncytiotrophoblast layer that mediates a wealth of essential functions to support and maintain a successful pregnancy. Furthermore, DEPs are found in placental macrophages and fetal endothelial cells, showing that some particles can overcome the syncytiotrophoblasts to reach the fetal capillaries. Few particles are also observed inside fetal microvessels.ConclusionsOverall, we show that DEPs accumulate in key cell types of the placental tissue and can cross the human placenta, although in limited amounts. These findings are crucial for risk assessment and protection of pregnant women and highlight the urgent need for further research on the direct and indirect placenta-mediated developmental toxicity of ambient particulates. This work received fnancial support from the Flemish Scientifc Research Foundation (Grant no 1150920N, G082317N, and 12P6819N) and the Swiss National Science Foundation (Grant no 31003A_179337 and IZSEZ0_193948). The detection equipment was funded by the Interuniversity Attraction Poles Program (P7/05) initiated by the Belgian Science Policy Ofce and the INCALO project (ERC-PoC). Acknowledgements The authors would like to acknowledge all participants of the study. In addition, we owe special thanks to the nurses, midwives, and doctors involved in placenta collection. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Airborne pollution particles have been shown to translocate from the mother’s lung to the fetal circulation but their placental-fetal distribution and internal tissue load remain poorly explored. Here, we investigated the placental-fetal distribution of diesel engine exhaust particles during gestation under controlled exposure conditions using a pregnant rabbit model. Pregnant dams were exposed by nose-only inhalation to either clean air (controls) or diluted and filtered diesel engine exhaust (1 mg/m3) for 2h/day, 5 days/week, from gestational day (GD) 3 to GD27. At GD28, placental and fetal tissues (i.e., heart, kidney, liver, lung and gonads) were collected for biometry and to study the accumulation of carbon particles (CPs) using white light generation by carbon particles under femtosecond pulsed laser illumination. CPs were detected in the placenta, fetal heart, kidney, liver, lung and gonads in significantly higher amounts compared with controls. By using multiple factor analysis, we were able to discriminate the diesel engine-exposed pregnant rabbits from the control group taking all variables related to fetoplacental biometry and CP load into consideration. Although our findings did not reveal a sex effect, a potential interaction effect might be present between exposure and fetal sex. The results confirm the translocation of maternally inhaled CPs from diesel engine exhaust to the placenta and the ability to reach fetal organs during late-stage pregnancy. The differential particle load in the fetal organs may contribute to the disruption of blood flow in the placenta with possible effects on fetoplacental biometry and placental function.

Background Maternal exposure to particulate air pollution during pregnancy has been linked to multiple adverse birth outcomes causing burden of disease later in the child's life. To date, there is a paucity of data on whether or not ambient particles can both reach and cross the human placenta to exert direct effects on fetal organ systems during gestation. Methods In this analysis, we used maternal-perinatal and fetal samples collected within the framework of two independent studies: the ENVIRONAGE (Environmental Influences on Ageing in Early Life) birth cohort of mothers giving birth at the East-Limburg Hospital in Genk, Belgium, and the SAFeR (Scottish Advanced Fetal Research) cohort of terminated, normally progressing pregnancies among women aged 16 years and older in Aberdeen and the Grampian region, UK. From the ENVIRONAGE study, we included 60 randomly selected mother-neonate pairs, excluding all mothers who reported that they ever smoked. From the SAFeR study, we included 36 fetuses of gestational age 7-20 weeks with cotinine concentrations indicative of non-smoking status. We used white light generation under femtosecond pulsed illumination to detect black carbon particles in samples collected at the maternal-fetal interface. We did appropriate validation experiments of all samples to confirm the carbonaceous nature of the identified particles. Findings We found evidence of the presence of black carbon particles in cord blood, confirming the ability of these particles to cross the placenta and enter the fetal circulation system. We also found a strong correlation (r >= 0.50; p < 0.0001) between the maternal-perinatal particle load (in maternal blood [n=60], term placenta [n=60], and cord blood [n=60]) and residential ambient black carbon exposure during pregnancy. Additionally, we found the presence of black carbon particles in first and second trimester tissues (fetal liver [n=36], lung [n=36], and brain [n=14]) of electively terminated and normally progressing pregnancies from an independent study. Interpretation We found that maternally inhaled carbonaceous air pollution particles can cross the placenta and then translocate into human fetal organs during gestation. These findings are especially concerning because this window of exposure is key to organ development. Further studies are needed to elucidate the mechanisms of particle translocation. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd. The ENVIRONAGE birth cohort was initiated by the European Research Council (ERC-2012-StG 310898) and received additional funding from the Flemish Scientific Research Foundation and Kom op Tegen Kanker (KoTK). The detection equipment was funded by the METHUSALEM Program and the INCALO project (ERC-PoC). We acknowledge the Flemish Scientific Research Foundation (FWO; 1150920N to EB and G082317N). The SAFeR study was funded by the UK Medical Research Council (MR/L010011/1 and MR/P011535/1) and the EU’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie project PROTECTED (grant agreement number 722634) and FREIA project (grant agreement number 825100) as well as by NHS Grampian Endowments grants (16/11/056, 17/034, 18/14, 19/029, and 20/031) to PAF. We thank the midwives from the maternity ward of the East-Limburg Hospital in Genk, Belgium, for coordinating and supporting the study at the ward. We thank the Advanced Optical Microscopy Centre for the maintenance of the microscopic instruments. Moreover, we thank our colleagues from the Centre for Environmental Sciences for their hard work in collecting and processing the samples for the ENVIRONAGE birth cohort. Additionally, we thank the NHS Grampian Research Nurses and NHS Grampian R&D for their tireless recruitment work for the SAFeR study. We thank the past and present SAFeR team for their hard work with the fetuses and placentae. Finally, we thank the NHS Grampian Biorepository for their oversight role in SAFeR and assistance in processing and preparation of tissue sections.

Particle transfer across the placenta has been suggested but to date, no direct evidence in real-life, human context exists. Here we report the presence of black carbon (BC) particles as part of combustion-derived particulate matter in human placentae using white-light generation under femtosecond pulsed illumination. BC is identified in all screened placentae, with an average (SD) particle count of 0.95 × 10

Particle transfer across the placenta has been suggested but to date, no direct evidence in real-life, human context exists. Here we report the presence of black carbon (BC) particles as part of combustion-derived particulate matter in human placentae using white-light generation under femtosecond pulsed illumination. BC is identified in all screened placentae, with an average (SD) particle count of 0.95 × 104 (0.66 × 104) and 2.09 × 104 (0.9 × 104) particles per mm3 for low and high exposed mothers, respectively. Furthermore, the placental BC load is positively associated with mothers’ residential BC exposure during pregnancy (0.63–2.42 µg per m3). Our finding that BC particles accumulate on the fetal side of the placenta suggests that ambient particulates could be transported towards the fetus and represents a potential mechanism explaining the detrimental health effects of pollution from early life onwards., Exposure to air pollution during pregnancy has been associated with impaired birth outcomes. Here, Bové et al. report evidence of black carbon particle deposition on the fetal side of human placentae, including at early stages of pregnancy, suggesting air pollution could affect birth outcome through direct effects on the fetus.

Fluorescent conjugated polymers formulated in nanoparticles show attractive properties to be used as bioimaging probes. However, their fluorescence brightness is generally limited by quenching phenomena due to interchain aggregation in the confined nanoparticle space. In this work, branched conjugated polymer networks are investigated as a way to enhance the photoluminescence quantum yield of the resulting conjugated polymer nanoparticles (CPNs). 1,3,5-Tribromobenzene and 2,2',7,7'-tetrabromo-9,9'-spirobifluorene are chosen as branching moieties and are added in 3 or 5 mol % to the poly(p-phenylene ethynylene) (PPE) conjugated polymer synthesis. Nanoparticles of all samples are prepared via the combined miniemulsion/solvent evaporation technique. The optical properties of the branched polymers in solution and in nanoparticle form are then compared to those of the linear PPE counterpart. The fluorescence quantum yield of the CPNs increases from 5 to 11% for the samples containing 1,3,5-tribromobenzene. Furthermore, when 5 mol % of either branching molecule is used, the one-photon fluorescence brightness doubles. The nanoparticles show low cytotoxicity in A549 human lung carcinoma cells up to a concentration of 100 μg/mL for 24 h. They also exhibit good particle uptake into cells and compatibility with two-photon imaging. ispartof: ACS BIOMATERIALS SCIENCE & ENGINEERING vol:5 issue:4 pages:1967-1977 ispartof: location:United States status: published

Fluorescent conjugated polymers formulated in nanoparticles show attractive properties to be used as bioimaging probes. However, their fluorescence brightness is generally limited by quenching phenomena due to interchain aggregation in the confined nanoparticle space. In this work, branched conjugated polymer networks are investigated as a way to enhance the photoluminescence quantum yield of the resulting conjugated polymer nanoparticles (CPNs). 1,3,5-Tribromobenzene and 2,2',7,7'-tetrabromo-9,9'-spirobifluorene are chosen as branching moieties and are added in 3 or 5 mol % to the poly(

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, we used multicolor single-molecule burst analysis microscopy to detect multiple biomarkers present on single EV. We classified the recorded signals and applied the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, we applied the method to assess both the purity and the inflammatory status of EV, and compared cell culture and plasma-derived EV isolated via different purification methods. We then applied this methodology to identify intercellular adhesion molecule-1 (ICAM-1) specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. Our methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.

Indoor plants can be used to monitor atmospheric particulates. Here, we report the label-free detection of combustion-derived particles (CDPs) on plants as a monitoring tool for indoor pollution. First, we measured the indoor CDP deposition on Atlantic ivy leaves (Hedera hibernica) using two-photon femtosecond microscopy. Subsequently, to prove its effectiveness for using it as a monitoring tool, ivy plants were placed near five different indoor sources. CDP particle area and number were used as output metrics. CDP values ranged between a median particle area of 0.45 x 10² to 1.35 x 10^4 mm² , and a median particle number of 0.10 x 10² to 1.42 x 10³ particles for the indoor sources: control (greenhouse) < milling machine < indoor smokers < wood stove < gas stove < laser printer. Our findings demonstrate that Atlantic ivy, combined with label-free detection, can be effectively used in indoor atmospheric monitoring studies. The authors thank Mr. E. Slenders for designing the analysis software in Matlab (Matlab R2017b (9.3.0.713579), MathWorks, Eindhoven, the Netherlands)

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.

Background In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs). Methods and results We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1 beta by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1 beta responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples. Conclusion The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells. This work was supported by the Fonds de la Recherche scientifique - FNRS [Convention de recherche ERA-NET - No R.50.17.16.F], the Flemish Scientific Research Foundation [FWO; HB: 12P6819N], the U.S.A. National Science Foundation [grant CBET-1530505], and the German BMBF (FKZ: 03XP0063). Lison, D (corresponding author), Catholic Univ Louvain, Louvain Ctr Toxicol & Appl Pharmacol LTAP, Inst Rech Expt & Clin IREC, Brussels, Belgium. dominique.lison@uclouvain.be

Fetal development is a crucial window of susceptibility in which exposure may lead to detrimental health outcomes at birth and later in life. The placenta serves as a gatekeeper between mother and fetus. Knowledge regarding the barrier capacity of the placenta for nanoparticles is limited, mostly due to technical obstacles and ethical issues. We systematically summarize and discuss the current evidence and define knowledge gaps concerning the maternal-fetal transport and fetoplacental accumulation of (ultra)fine particles and nanoparticles. We included 73 studies on placental translocation of particles, of which 21 in vitro/ex vivo studies, 50 animal studies, and 2 human studies on transplacental particle transfer. This systematic review shows that (i) (ultra)fine particles and engineered nanoparticles can bypass the placenta and reach fetal units as observed for all the applied models irrespective of the species origin (i.e., rodent, rabbit, or human) or the complexity (i.e., in vitro, ex vivo, or in vivo), (ii) particle size, particle material, dose, particle dissolution, gestational stage of the model, and surface composition influence maternal-fetal translocation, and (iii) no simple, standardized method for nanoparticle detection and/or quantification in biological matrices is available to date. Existing evidence, research gaps, and perspectives of maternal-fetal particle transfer are highlighted.

Boron dipyrromethene (BODIPY) dyes represent a particular class within the broad array of potential photosensitizers. Their highly fluorescent nature opens the door for theragnostic applications, combining imaging and therapy using a single, easily synthesized chromophore. However, near-infrared absorption is strongly desired for photodynamic therapy to enhance tissue penetration. Furthermore, singlet oxygen should preferentially be generated without the incorporation of heavy atoms, as these often require additional synthetic efforts and/or afford dark cytotoxicity. Solutions for both problems are known, but have never been successfully combined in one simple BODIPY material. Here, we present a series of compact BODIPY-acridine dyads, active in the phototherapeutic window and showing balanced brightness and phototoxic power. Although the donor-acceptor design was envisioned to introduce a charge transfer state to assist in intersystem crossing, quantum-chemical calculations refute this. Further photophysical investigations suggest the presence of exciplex states and their involvement in singlet oxygen formation. The authors thank Hasselt University and the University of Namur for continuing financial support (PhD scholarships J.D. and T.C.). M.K., B.C., and W.M. thank the Research Foundation-Flanders (FWO) for support through project G087718N, Hercules project GOH3816NAUHL, and the Scientific Research Community (WOG) "Supramolecular Chemistry and Materials" (W000620N). The calculations were performed on the computers of the "Consortium des equipements de Calcul Intensif" (CECI) (http://www.ceci-hpc.be), including those of the "UNamur Technological Platform of High-Performance Computing" (PTCI) (http://www.ptci.unamur.be), for which we gratefully acknowledge the financial support from the FNRS-FRFC, the Walloon Region, and the University of Namur (Conventions No. 2.5020.11, GEQ U.G006.15, U.G018.19, 1610468, and RW/GEQ2016). Maes, W (corresponding author), UHasselt Hasselt Univ, Inst Mat Res IMO, Design & Synth Organ Semicond DSOS, B-3590 Diepenbeek, Belgium ; IMEC, Associated Lab IMOMEC, Wetenschapspk 1, B-3590 Diepenbeek, Belgium. wouter.maes@uhasselt.be

Two-photon excitation (2PE) laser scanning microscopy is the imaging modality of choice when one desires to work with thick biological samples. However, its spatial resolution is poor, below confocal laser scanning microscopy. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction method, is shown to enhance the effective resolution, as well as the overall image quality of 2PE microscopy. With our adaptive pixel reassignment procedure similar to 1.6 times resolution increase is maintained deep into thick semi-transparent samples. The integration of Fourier ring correlation based semi-blind deconvolution is shown to further enhance the effective resolution by a factor of similar to 2 - and automatic background correction is shown to boost the image quality especially in noisy images. Most importantly, our 2PE-ISM implementation requires no calibration measurements or other input from the user, which is an important aspect in terms of day-to-day usability of the technique. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement H2020 Marie Sklodowska-Curie Actions (AdaptiveSTED, No. 794531); FondsWetenschappelijk Onderzoek (G092915, V429717N); European Research Council (Bright Eyes, No. 818699). Vicidomini, G (corresponding author), Ist Italiano Tecnol, Mol Microscopy & Spect, Genoa, Italy giuseppe.vicidomini@iit.it

We propose a straightforward implementation of two-photon image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction algorithm is shown to dramatically improve the optical resolution of two-photon imaging, in various test samples. We show how our computational ISM approach is able to adapt to changing imaging conditions, thus ensuring optimal image quality. We also show how our recently introduced blind deconvolution approaches can be integrated into the image reconstruction workflow to further improve the image quality.

Conjugated polymers are versatile bio-imaging probes as their optical properties can be readily fine-tuned. In this manuscript, fluorescent conjugated polymer nanoparticles are fabricated using three different poly(p-phenylene ethynylene) (PPE) derivatives. The polymers have the same backbone but carry different side chains, i.e. regular octyloxy substituents, half of the octyloxy chains azide terminated, or azide functionalized tetraethylene glycol (TEG) moieties. The azide groups are specifically chosen to allow coupling of (bio)molecules to the surface of the particles using straightforward azide-alkyne click reactions, enabling different bioconjugation and targeting strategies. The influence of the functionalization pattern on the size and optical properties of the nanoparticles is studied using transmission electron microscopy, dynamic light scattering, UV-Vis absorption and fluorescence spectroscopy. The polymer containing the azide functionalized TEG chains affords larger particles, which can be attributed to hydration of the outer layer of the more hydrophilic polymer particles. However, this does not impact the fluorescence quantum yield. The two azide functionalized PPE particles exhibit the highest quantum yields (13%). Despite the presence of azide groups on two of the three materials, all particles are biocompatible and taken up by A549 human lung carcinoma cells. A proof of concept click reaction was performed as well.

Rationale: Ambient air pollution, including black carbon, entails a serious public health risk because of its carcinogenic potential and as climate pollutant. To date, an internal exposure marker for black carbon particles that have cleared from the systemic circulation into the urine does not exist. Objectives: To develop and validate a novel method to measure black carbon particles in a label-free way in urine. Methods: We detected urinary carbon load in 289 children (aged 9–12 yr) using white-light generation under femtosecond pulsed laser illumination. Children’s residential black carbon concentrations were estimated based on a high-resolution spatial temporal interpolation method. Measurements and Main Results: We were able to detect urinary black carbon in all children, with an overall average (SD) of 98.2 × 105 (29.8 × 105) particles/ml. The urinary black carbon load was positively associated with medium-term to chronic (1 mo or more) residential black carbon exposure: +5.33 × 105 particles/ml higher carbon load (95% confidence interval, 1.56 × 105 to 9.10 × 105 particles/ml) for an interquartile range increment in annual residential black carbon exposure. Consistently, children who lived closer to a major road (≤160 m) had higher urinary black carbon load (6.93 × 105 particles/ml; 95% confidence interval, 0.77 × 105 to 13.1 × 105). Conclusions: Urinary black carbon mirrors the accumulation of medium-term to chronic exposure to combustion-related air pollution. This specific biomarker reflects internal systemic black carbon particles cleared from the circulation into the urine, allowing investigators to unravel the complexity of particulate-related health effects. Funded by the European Research Council (ERC-2012-StG 310898), the Interuniversity Attraction Poles Program (P7/05) initiated by the Belgian Science Policy Office, and the Flemish Scientific Research Foundation (G082113/11ZB115N/12R6315N/G073315N).

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (similar to 7 days) to elderly (similar to 38 months) rabbits were monitored in intact (n(tendons) = 24) and microtome sectioned (ntendons = 11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Lambda) started at very low values (A = 2.0 +/- 0.6 mu m, Lambda = 19 +/- 4 mu m) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development. The project was mainly supported by the Grant Agency of Charles University (grant no. GAUK 956213), Czech Science Foundation, grant no. 16-14758S, by the Ministry of Health of the Czech Republic, grant no. 16-28637A, by the Ministry of Education, Youth and Sports within National Sustainability Programme I, project no. LO1309, by MEYS (LM2015062 Czech-BioImaging), by ERDF OPPK Brain-View CZ.2.16/3.1.00/21544, and D.H. acknowledges the support from the Flemish Government and Hasselt University for a research stay at Hasselt University.

Despite the spontaneous regenerative capacity of the peripheral nervous system, large gap peripheral nerve injuries (PNIs) require bridging strategies. The limitations and suboptimal results obtained with autografts or hollow nerve conduits in the clinic urge the need for alternative treatments. Recently, we have described promising neuroregenerative capacities of Schwann cells derived from differentiated human dental pulp stem cells (d-hDPSCs) in vitro. Here, we extended the in vitro assays to show the pro-angiogenic effects of d-hDPSCs, such as enhanced endothelial cell proliferation, migration and differentiation. In addition, for the first time we evaluated the performance of d-hDPSCs in an in vivo rat model of PNI. Eight weeks after transplantation of NeuraWrap™ conduits filled with engineered neural tissue (EngNT) containing aligned d-hDPSCs in 15-mm rat sciatic nerve defects, immunohistochemistry and ultrastructural analysis revealed ingrowing neurites, myelinated nerve fibres and blood vessels along the construct. Although further research is required to optimize the delivery of this EngNT, our findings suggest that d-hDPSCs are able to exert a positive effect in the regeneration of nerve tissue in vivo. Copyright © 2017 John Wiley & Sons, Ltd.

CD34+ hematopoietic progenitor cells (HPCs) offer great opportunities to develop new treatments for numerous malignant and non-malignant diseases. Nanoparticle (NP)-based strategies can further enhance this potential, and therefore a thorough understanding of the loading behavior of HPCs towards NPs is essential for a successful application. The present study focusses on the interaction kinetics of 40 nm sized carboxylated polystyrene (PS) NPs with HPCs. Interestingly, a transient association of the NPs with HPCs is observed, reaching a maximum within 1 hour and declining afterwards. This behavior is not seen in dendritic cells (CD34-DCs) differentiated from HPCs, which display a monotonic increase in NP load. We demonstrate that this transient interaction requires an energy-dependent cellular process, suggesting active loading and release of NPs by HPCs. This novel observation offers a unique approach to transiently equip HPCs. A simple theoretical approach modeling the kinetics of NP loading and release is presented, contributing to a framework of describing this phenomenon.

Conjugated polymer nanoparticles exhibit very interesting properties for use as bio-imaging agents. In this paper, we report the synthesis of PCDTBT (poly([9-(1&rsquo, octylnonyl)-9H-carbazole-2,7-diyl]-2,5-thiophenediyl-2,1,3-benzothiadiazole-4,7-diyl-2,5-thiophene-diyl)) nanoparticles of varying sizes using the mini-emulsion and emulsion/solvent evaporation approach. The effect of the size of the particles on the optical properties is investigated using UV-Vis absorption and fluorescence emission spectroscopy. It is shown that PCDTBT nanoparticles have a fluorescence emission maximum around 710 nm, within the biological near-infrared &ldquo, optical window&rdquo, The photoluminescence quantum yield shows a characteristic trend as a function of size. The particles are not cytotoxic and are taken up successfully by human lung cancer carcinoma A549 cells. Irrespective of the size, all particles show excellent fluorescent brightness for bioimaging. The fidelity of the particles as fluorescent probes to study particle dynamics in situ is shown as a proof of concept by performing raster image correlation spectroscopy. Combined, these results show that PCDTBT is an excellent candidate to serve as a fluorescent probe for near-infrared bio-imaging.

Microtubules are a vital component of the cell’s cytoskeleton and their organization is crucial for healthy cell functioning. The use of label-free SH imaging of microtubules remains limited, as sensitive detection is required and the true molecular origin and main determinants required to generate SH from microtubules are not fully understood. Using advanced correlative imaging techniques, we identified the determinants of the microtubule-dependent SH signal. Microtubule polarity, number and organization determine SH signal intensity in biological samples. At the molecular level, we show that the GTP-bound tubulin dimer conformation is fundamental for microtubules to generate detectable SH signals. We show that SH imaging can be used to study the effects of microtubule-targeting drugs and proteins and to detect changes in tubulin conformations during neuronal maturation. Our data provide a means to interpret and use SH imaging to monitor changes in the microtubule network in a label-free manner., Microtubules (MTs) are well-studied cytoskeleton components, but have primarily been investigated using fixation or invasive techniques. Here, the authors use label-free second harmonic (SH) fluorescence and correlative light electron microscopy to pinpoint determinants required for SH from MTs.

Silver nanoparticles (AgNPs) currently belong to the most commercialized nanomaterials, used in both consumer products and medical applications. Despite its omnipresence, in-depth knowledge on the potential toxicity of nanosilver is still lacking, especially for developing organisms. Research on vertebrates is limited due to ethical concerns, and planarians are an ideal invertebrate model to study the effects of AgNPs on stem cells and developing tissues in vivo, as regeneration mimics development by triggering massive stem cell proliferation. Our results revealed a strong interference of AgNPs with tissue- and neuroregeneration which was related to an altered stem cell cycle. The presence of a PVP-coating significantly influenced toxicity outcomes, leading to elevated DNA-damage and decreased stem cell proliferation. Non-coated AgNPs had an inhibiting effect on stem cell and early progeny numbers. Overall, regenerating tissues were more sensitive to AgNP toxicity, and careful handling and appropriate decision making is needed in AgNP applications for healing and developing tissues. We emphasize on the importance of AgNP characterization, as we showed that changes in physicochemical properties influence toxicity. This study was financially supported by Bijzonder Onderzoeksfonds Hasselt University; Fonds Wetenschappelijk Onderzoek Onderzoek Vlaanderen; Province of Limburg and Vlaamse Instelling voor Technologisch Onderzoek.

Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracting the mobility, concentration, and stoichiometry of diffusing fluorescent molecules from confocal image stacks. The method works by calculating a spatial correlation function for each image and analyzing the average of those by model fitting. Rules of thumb exist for RICS image acquisitioning, yet a rigorous theoretical approach to predict the accuracy and precision of the recovered parameters has been lacking. We outline explicit expressions to reveal the dependence of RICS results on experimental parameters. In terms of imaging settings, we observed that a twofold decrease of the pixel size, e.g., from 100 to 50 nm, decreases the error on the translational diffusion constant (D) between three- and fivefold. For D = 1 μm2 s−1, a typical value for intracellular measurements, ∼25-fold lower mean-squared relative error was obtained when the optimal scan speed was used, although more drastic improvements were observed for other values of D. We proposed a slightly modified RICS calculation that allows correcting for the significant bias of the autocorrelation function at small (≪50 × 50 pixels) sizes of the region of interest. In terms of sample properties, at molecular brightness E = 100 kHz and higher, RICS data quality was sufficient using as little as 20 images, whereas the optimal number of frames for lower E scaled pro rata. RICS data quality was constant over the nM–μM concentration range. We developed a bootstrap-based confidence interval of D that outperformed the classical least-squares approach in terms of coverage probability of the true value of D. We validated the theory via in vitro experiments of enhanced green fluorescent protein at different buffer viscosities. Finally, we outline robust practical guidelines and provide free software to simulate the parameter effects on recovery of the diffusion coefficient. Funding details: KU Leuven, C14/16/053; Funding details: Fonds Wetenschappelijk Onderzoek, FWO; Funding details: Knut och Alice Wallenbergs Stiftelse; Funding text 1: Dr. Kris Janssen (Chemistry, KU Leuven) is thanked for programming the acquisitioning software of the homebuilt microscope. Johan Hofkens (Molecular Imaging and Photonics, KU Leuven) is gratefully acknowledged for the usage of his imaging facilities. Financial support from the Swedish Foundation for Strategic Research and Knut and Alice Wallenberg Foundation is highly appreciated. J.H. acknowledges the KU Leuven for funding ( C14/16/053 ). V.L. acknowledges the UHasselt Bijzonder Onderzoeksfonds fund ( BOF17DOC11 ) for a PhD scholarship. Guillermo Solís Fernández is grateful for a PhD scholarship from the Research Foundation Flanders.

Characterization of the membrane phases is a crucial task in cell biology. Cells differ in composition of the lipids and consequently in adopted phases. The phases can be discriminated based upon lipid ordering and molecular diffusion and their identification could be used for characterization of cell membranes. Here we used molecular dynamics (MD) simulations to study the behavior of the fluorescent reporter molecule diphenylhexatriene (DPH) in different lipid phases – liquid disordered (Ld), liquid ordered (Lo), and solid ordered (So) composed of phosphatidylcholines (Ld and So) or a sphingomyelin/cholesterol (SM/Chol) mixture (Lo). To the best of our knowledge, this is the first simulation of DPH in Lo SM/Chol and So DPPC membranes. For the considered membrane compositions DPH is mostly oriented parallel to lipid tails. In the Lo phase we observed a significant fraction of DPH positioned in between membrane leaflets, which agrees with experimental findings, but which has not been observed in previous MD simulations of DPH in phosphatidylcholine membranes. Further, we calculated rotational autocorrelation functions (ROTACF) from our MD simulations in order to model the time-resolved fluorescence anisotropy decay. We observed that order parameters 〈P2〉 and 〈P4〉 are sufficient to fully describe the orientation distribution of DPH. We analyzed the ROTACFs by a so-called general model for the time-resolved fluorescence anisotropy [W. van der Meer et al., Biophys. J., 1984, 46, 515] and observed an overestimation of 〈P4〉. We suggest a rescaling of the recovered 〈P4〉 yielding an orientation distribution of DPH close to the one observed in our MD simulations.