Accumulation of Ambient Black Carbon Particles Within Key Memory-Related Brain Regions.

Importance: Ambient air pollution is a worldwide problem, not only related to respiratory and cardiovascular diseases but also to neurodegenerative disorders. Different pathways on how air pollutants could affect the brain are already known, but direct evidence of the presence of ambient particles (or nanoparticles) in the human adult brain is limited. Objective: To examine whether ambient black carbon particles can translocate to the brain and observe their biodistribution within the different brain regions. Design, Setting, and Participants: In this case series a label-free and biocompatible detection technique of nonincandescence-related white light generation was used to screen different regions of biobanked brains of 4 individuals from Belgium with neuropathologically confirmed Alzheimer disease for the presence of black carbon particles. The selected biological specimens were acquired and subsequently stored in a biorepository between April 2013 and April 2017. Black carbon measurements and data analysis were conducted between June 2020 and December 2022. Main Outcomes and Measures: The black carbon load was measured in various human brain regions. A Kruskal-Wallis test was used to compare black carbon loads across these regions, followed by Dunn multiple comparison tests. Results: Black carbon particles were directly visualized in the human brain of 4 individuals (3 women [75%]; mean [SD] age, 86 [13] years). Screening of the postmortem brain regions showed a significantly higher median (IQR) number of black carbon particles present in the thalamus (433.6 [289.5-540.2] particles per mm3), the prefrontal cortex including the olfactory bulb (420.8 [306.6-486.8] particles per mm3), and the hippocampus (364.7 [342.0-448.7] particles per mm3) compared with the cingulate cortex (192.3 [164.2-277.5] particles per mm3), amygdala (217.5 [147.3-244.5] particles per mm3), and the superior temporal gyrus (204.9 [167.9-236.8] particles per mm3). Conclusions and Relevance: This case series provides evidence that ambient air pollution particles are able to translocate to the human brain and accumulate in multiple brain regions involved in cognitive functioning. This phenomenon may contribute to the onset and development of neurodegenerative disorders.

Generalized polarization and time-resolved fluorescence provide evidence for different populations of Laurdan in lipid vesicles.

The solvatochromic dye Laurdan is widely used in sensing the lipid packing of both model and biological membranes. The fluorescence emission maximum shifts from about 440 nm (blue channel) in condensed membranes (So) to about 490 nm (green channel) in the liquid-crystalline phase (Lα). Although the fluorescence intensity based generalized polarization (GP) is widely used to characterize lipid membranes, the fluorescence lifetime of Laurdan, in the blue and the green channel, is less used for that purpose. Here we explore the correlation between GP and fluorescence lifetimes by spectroscopic measurements on the So and Lα phases of large unilamellar vesicles of DMPC and DPPC. A positive correlation between GP and the lifetimes is observed in each of the optical channels for the two lipid phases. Microfluorimetric determinations on giant unilamellar vesicles of DPPC and DOPC at room temperature are performed under linearly polarized two-photon excitation to disentangle possible subpopulations of Laurdan at a scale below the optical resolution. Fluorescence intensities, GP and fluorescence lifetimes depend on the angle between the orientation of the linear polarization of the excitation light and the local normal to the membrane of the optical cross-section. This angular variation depends on the lipid phase and the emission channel. GP and fluorescence intensities in the blue and green channel in So and in the blue channel in Lα exhibit a minimum near 90o. Surprisingly, the intensity in the green channel in Lα reaches a maximum near 90o. The fluorescence lifetimes in the two optical channels also reach a pronounced minimum near 90o in So and Lα, apart from the lifetime in the blue channel in Lα where the lifetime is short with minimal angular variation. To our knowledge, these experimental observations are the first to demonstrate the existence of a bent conformation of Laurdan in lipid membranes, as previously suggested by molecular dynamics calculations.

Ambient black carbon particles in human ovarian tissue and follicular fluid.

Evidence indicates a link between exposure to ambient air pollution and decreased female fertility. The ability of air pollution particles to reach human ovarian tissue and follicles containing the oocytes in various maturation stages has not been studied before. Particulate translocation might be an essential step in explaining reproductive toxicity and assessing associated risks. Here, we analysed the presence of ambient black carbon particles in (i) follicular fluid samples collected during ovum pick-up from 20 women who underwent assisted reproductive technology treatment and (ii) adult human ovarian tissue from 5 individuals. Follicular fluid and ovarian tissue samples were screened for the presence of black carbon particles from ambient air pollution using white light generation by carbonaceous particles under femtosecond pulsed laser illumination. We detected black carbon particles in all follicular fluid (n = 20) and ovarian tissue (n = 5) samples. Black carbon particles from ambient air pollution can reach the ovaries and follicular fluid, directly exposing the ovarian reserve and maturing oocytes. Considering the known link between air pollution and decreased fertility, the impact of such exposure on oocyte quality, ovarian ageing and fertility needs to be clarified urgently.

Pre-admission ambient air pollution and blood soot particles predict hospitalisation outcomes in COVID-19 patients.

BACKGROUND: Air pollution exposure is one of the major risk factors for aggravation of respiratory diseases. We investigated whether exposure to air pollution and accumulated black carbon (BC) particles in blood were associated with coronavirus disease 2019 (COVID-19) disease severity, including the risk for intensive care unit (ICU) admission and duration of hospitalisation. METHODS: From May 2020 until March 2021, 328 hospitalised COVID-19 patients (29% at intensive care) were recruited from two hospitals in Belgium. Daily exposure levels (from 2016 to 2019) for particulate matter with aerodynamic diameter <2.5 µm and <10 µm (PM2.5 and PM10, respectively), nitrogen dioxide (NO2) and BC were modelled using a high-resolution spatiotemporal model. Blood BC particles (internal exposure to nano-sized particles) were quantified using pulsed laser illumination. Primary clinical parameters and outcomes included duration of hospitalisation and risk of ICU admission. RESULTS: Independent of potential confounders, an interquartile range (IQR) increase in exposure in the week before admission was associated with increased duration of hospitalisation (PM2.5 +4.13 (95% CI 0.74-7.53) days, PM10 +4.04 (95% CI 1.24-6.83) days and NO2 +4.54 (95% CI 1.53-7.54) days); similar effects were observed for long-term NO2 and BC exposure on hospitalisation duration. These effect sizes for an IQR increase in air pollution on hospitalisation duration were equivalent to the effect of a 10-year increase in age on hospitalisation duration. Furthermore, for an IQR higher blood BC load, the OR for ICU admission was 1.33 (95% CI 1.07-1.65). CONCLUSIONS: In hospitalised COVID-19 patients, higher pre-admission ambient air pollution and blood BC levels predicted adverse outcomes. Our findings imply that air pollution exposure influences COVID-19 severity and therefore the burden on medical care systems during the COVID-19 pandemic.

Ambient black carbon reaches the kidneys.

BACKGROUND: Ultrafine particles, including black carbon (BC), can reach the systemic circulation and therefore may distribute to distant organs upon inhalation. The kidneys may be particularly vulnerable to the adverse effects of BC exposure due to their filtration function. OBJECTIVES: We hypothesized that BC particles reach the kidneys via the systemic circulation, where the particles may reside in structural components of kidney tissue and impair kidney function. METHODS: In kidney biopsies from 25 transplant patients, we visualized BC particles using white light generation under femtosecond-pulsed illumination. The presence of urinary kidney injury molecule-1 (KIM-1) and cystatin c (CysC) were evaluated with ELISA. We assessed the association between internal and external exposure matrices and urinary biomarkers using Pearson correlation and linear regression models. RESULTS: BC particles could be identified in all biopsy samples with a geometric mean (5th, 95th percentile) of 1.80 × 103 (3.65 × 102, 7.50 × 103) particles/mm3 kidney tissue, predominantly observed in the interstitium (100 %) and tubules (80 %), followed by the blood vessels and capillaries (40 %), and the glomerulus (24 %). Independent from covariates and potential confounders, we found that each 10 % higher tissue BC load resulted in 8.24 % (p = 0.03) higher urinary KIM-1. In addition, residential proximity to a major road was inversely associated with urinary CysC (+10 % distance: -4.68 %; p = 0.01) and KIM-1 (+10 % distance: -3.99 %; p < 0.01). Other urinary biomarkers, e.g., the estimated glomerular filtration rate or creatinine clearance showed no significant associations. DISCUSSION AND CONCLUSION: Our findings that BC particles accumulate near different structural components of the kidney represent a potential mechanism explaining the detrimental effects of particle air pollution exposure on kidney function. Furthermore, urinary KIM-1 and CysC show potential as air pollution-induced kidney injury biomarkers for taking a first step in addressing the adverse effects BC might exert on kidney function.

Placental-fetal distribution of carbon particles in a pregnant rabbit model after repeated exposure to diluted diesel engine exhaust.

BACKGROUND: Airborne pollution particles have been shown to translocate from the mother's lung to the fetal circulation, but their distribution and internal placental-fetal tissue load remain poorly explored. Here, we investigated the placental-fetal load and 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 2 h/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 presence of carbon particles (CPs) using white light generation by carbonaceous particles under femtosecond pulsed laser illumination. RESULTS: CPs were detected in the placenta, fetal heart, kidney, liver, lung and gonads in significantly higher amounts in exposed rabbits compared with controls. Through 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. Our findings did not reveal a sex effect, yet a potential interaction effect might be present between exposure and fetal sex. CONCLUSIONS: The results confirmed the translocation of maternally inhaled CPs from diesel engine exhaust to the placenta which could be detected in fetal organs during late-stage pregnancy. The exposed can be clearly discriminated from the control group with respect to fetoplacental biometry and CP load. The differential particle load in the fetal organs may contribute to the effects on fetoplacental biometry and to the malprogramming of the fetal phenotype with long-term effects later in life.

Accumulation of Black Carbon Particles in Placenta, Cord Blood, and Childhood Urine in Association with the Intestinal Microbiome Diversity and Composition in Four- to Six-Year-Old Children in the ENVIRONAGE Birth Cohort.

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.

Association of urinary and ambient black carbon, and other ambient air pollutants with risk of prediabetes and metabolic syndrome in children and adolescents.

The effects of exposure to black carbon (BC) on various diseases remains unclear, one reason being potential exposure misclassification following modelling of ambient air pollution levels. Urinary BC particles may be a more precise measure to analyze the health effects of BC. We aimed to assess the risk of prediabetes and metabolic syndrome (MetS) in relation to urinary BC particles and ambient BC and to compare their associations in 5453 children from IDEFICS/I. Family cohort. We determined the amount of BC particles in urine using label-free white-light generation under femtosecond pulsed laser illumination. We assessed annual exposure to ambient air pollutants (BC, PM2.5 and NO2) at the place of residence using land use regression models for Europe, and we calculated the residential distance to major roads (≤250 m vs. more). We analyzed the cross-sectional relationships between urinary BC and air pollutants (BC, PM2.5 and NO2) and distance to roads, and the associations of all these variables to the risk of prediabetes and MetS, using logistic and linear regression models. Though we did not observe associations between urinary and ambient BC in overall analysis, we observed a positive association between urinary and ambient BC levels in boys and in children living ≤250 m to a major road compared to those living >250 m away from a major road. We observed a positive association between log-transformed urinary BC particles and MetS (ORper unit increase = 1.72, 95% CI = 1.21; 2.45). An association between ambient BC and MetS was only observed in children living closer to a major road. Our findings suggest that exposure to BC (ambient and biomarker) may contribute to the risk of MetS in children. By measuring the internal dose, the BC particles in urine may have additionally captured non-residential sources and reduced exposure misclassification. Larger studies, with longitudinal design including measurement of urinary BC at multiple time-points are warranted to confirm our findings.

Black carbon particles in human breast milk: assessing infant's exposure.

Background/Aim: Human breast milk is the recommended source of nutrition for infants due to its complex composition and numerous benefits, including a decline in infection rates in childhood and a lower risk of obesity. Hence, it is crucial that environmental pollutants in human breast milk are minimized. Exposure to black carbon (BC) particles has adverse effects on health; therefore, this pilot study investigates the presence of these particles in human breast milk. Methods: BC particles from ambient exposure were measured in eight human breast milk samples using a white light generation under femtosecond illumination. The carbonaceous nature of the particles was confirmed with BC fingerprinting. Ambient air pollution exposures (PM2.5, PM10, and NO2) were estimated using a spatial interpolation model based on the maternal residential address. Spearman rank correlation coefficients were obtained to assess the association between human breast milk's BC load and ambient air pollution exposure. Results: BC particles were found in all human breast milk samples. BC loads in human breast milk were strongly and positively correlated with recent (i.e., 1 week) maternal residential NO2 (r = 0.79; p = 0.02) exposure and medium-term (i.e., 1 month) PM2.5 (r = 0.83; p = 0.02) and PM10 (r = 0.93; p = 0.002) exposure. Conclusion: For the first time, we showed the presence of BC particles in human breast milk and found a robust association with ambient air pollution concentrations. Our findings present a pioneering insight into a novel pathway through which combustion-derived air pollution particles can permeate the delicate system of infants.

Maternal exposure to ambient black carbon particles and their presence in maternal and fetal circulation and organs: an analysis of two independent population-based observational studies.

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. FUNDING: European Research Council, Flemish Scientific Research Foundation, Kom op Tegen Kanker, UK Medical Research Council, and EU Horizon 2020.

Label-free detection of uptake, accumulation, and translocation of diesel exhaust particles in ex vivo perfused human placenta.

BACKGROUND: Pregnant 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. RESULTS: Here, 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. CONCLUSIONS: Overall, 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.

Femtosecond pulsed laser microscopy: a new tool to assess the in vitro delivered dose of carbon nanotubes in cell culture experiments.

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-1ß 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-1ß 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.

Unsupervised Machine Learning-Based Clustering of Nanosized Fluorescent Extracellular Vesicles.

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.

Translocation of (ultra)fine particles and nanoparticles across the placenta; a systematic review on the evidence of in vitro, ex vivo, and in vivo studies.

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.

Monitoring indoor exposure to combustion-derived particles using plants.

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 × 102 to 1.35 × 104 µm2, and a median particle number of 0.10 × 102 to 1.42 × 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.

Two-photon image-scanning microscopy with SPAD array and blind image reconstruction.

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 ∼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 ∼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.

Near-Infrared BODIPY-Acridine Dyads Acting as Heavy-Atom-Free Dual-Functioning Photosensitizers.

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.

Induction and recovery of CpG site specific methylation changes in human bronchial cells after long-term exposure to carbon nanotubes and asbestos.

INTRODUCTION: Inhalation of asbestos induces lung cancer via different cellular mechanisms. Together with the increased production of carbon nanotubes (CNTs) grows the concern about adverse effects on the lungs given the similarities with asbestos. While it has been established that CNT and asbestos induce epigenetic alterations, it is currently not known whether alterations at epigenetic level remain stable after withdrawal of the exposure. Identification of DNA methylation changes after a low dose of CNT and asbestos exposure and recovery can be useful to determine the fibre/particle toxicity and adverse outcome. METHODS: Human bronchial epithelial cells (16HBE) were treated with a low and non-cytotoxic dose (0.25 µg/ml) of multi-walled carbon nanotubes (MWCNTs-NM400) or single-walled carbon nanotubes (SWCNTs-SRM2483) and 0.05 µg/ml amosite (brown) asbestos for the course of four weeks (sub-chronic exposure). After this treatment, the cells were further incubated (without particle/fibre) for two weeks, allowing recovery from the exposure (recovery period). Nuclear depositions of the CNTs were assessed using femtosecond pulsed laser microscopy in a label-free manner. DNA methylation alterations were analysed using microarrays that assess more than 850 thousand CpG sites in the whole genome. RESULTS: At non-cytotoxic doses, CNTs were noted to be incorporated with in the nucleus after a four weeks period. Exposure to MWCNTs induced a single hypomethylation at a CpG site and gene promoter region. No change in DNA methylation was observed after the recovery period for MWCNTs. Exposure to SWCNTs or amosite induced hypermethylation at CpG sites after sub-chronic exposure which may involve in 'transcription factor activity' and 'sequence-specific DNA binding' gene ontologies. After the recovery period, hypermethylation and hypomethylation were noted for both SWCNTs and amosite. Hippocalcinlike 1 (HPCAL1), protease serine 3 (PRSS3), kallikrein-related peptidase 3 (KLK3), kruppel like factor 3 (KLF3) genes were hypermethylated at different time points in either SWCNT-exposed or amosite-exposed cells. CONCLUSION: These results suggest that the specific SWCNT (SRM2483) and amosite fibres studied induce hypo- or hypermethylation on CpG sites in DNA after very low-dose exposure and recovery period. This effect was not seen for the studied MWCNT (NM400).

Raster Image Correlation Spectroscopy Performance Evaluation.

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.