NKCC1 inhibition reduces periaxonal swelling, increases white matter sparing, and improves neurological recovery after contusive SCI.

Ultrastructural studies of contusive spinal cord injury (SCI) in mammals have shown that the most prominent acute changes in white matter are periaxonal swelling and separation of myelin away from their axon, axonal swelling, and axonal spheroid formation. However, the underlying cellular and molecular mechanisms that cause periaxonal swelling and the functional consequences are poorly understood. We hypothesized that periaxonal swelling and loss of connectivity between the axo-myelinic interface impedes neurological recovery by disrupting conduction velocity, and glial to axonal trophic support resulting in axonal swelling and spheroid formation. Utilizing in vivo longitudinal imaging of Thy1YFP+ axons and myelin labeled with Nile red, we reveal that periaxonal swelling significantly increases acutely following a contusive SCI (T13, 30 kdyn, IH Impactor) versus baseline recordings (laminectomy only) and often precedes axonal spheroid formation. In addition, using longitudinal imaging to determine the fate of myelinated fibers acutely after SCI, we show that ∼73% of myelinated fibers present with periaxonal swelling at 1 h post SCI and âˆ¼ 51% of those fibers transition to axonal spheroids by 4 h post SCI. Next, we assessed whether cation-chloride cotransporters present within the internode contributed to periaxonal swelling and whether their modulation would increase white matter sparing and improve neurological recovery following a moderate contusive SCI (T9, 50 kdyn). Mechanistically, activation of the cation-chloride cotransporter KCC2 did not improve neurological recovery and acute axonal survival, but did improve chronic tissue sparing. In distinction, the NKKC1 antagonist bumetanide improved neurological recovery, tissue sparing, and axonal survival, in part through preventing periaxonal swelling and disruption of the axo-myelinic interface. Collectively, these data reveal a novel neuroprotective target to prevent periaxonal swelling and improve neurological recovery after SCI.

Intracellular flow cytometric lipid analysis - a multiparametric system to assess distinct lipid classes in live cells.

The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content - intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue fluorescence, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric spanning-tree progression analysis for density-normalized events (SPADE) analysis of hepatic cellular lipid distribution, including vitamin A autofluorescence, is presented. This flow cytometric system allows for the rapid, inexpensive and non-destructive identification of lipid content, and highlights the differences in lipid biology between cell types by imaging and flow cytometry.

Ruthenium complexes bearing nile red chromophore and one of its derivative: Theoretical evaluation of PDT-related properties.

The outcomes of DFT-based calculations are here reported to assess the applicability of two synthesized polypyridyl Ru(II) complexes, bearing ethynyl nile red (NR) on a bpy ligand, and two analogues, bearing modified-NR, in photodynamic therapy. The absorption spectra, together with the non-radiative rate constants for the S1 - Tn intersystem crossing transitions, have been computed for this purpose. Calculations evidence that the structural modification on the chromophore destabilizes the HOMO of the complexes thus reducing the H-L gap and, consequently, red shifting the maximum absorption wavelength within the therapeutic window, up to 620 nm. Moreover, the favored ISC process from the bright state involves the triplet state closest in energy, which is also characterized by the highest SOC value and by the involvement of the whole bpy ligand bearing the chromophore in delocalising the unpaired electrons. These outcomes show that the photophysical behavior of the complexes is dominated by the chromophore.

Nile red staining for rapid screening of plastic-suspect particles in edible seafood tissues.

Concerns regarding microplastic (MP) contamination in aquatic ecosystems and its impact on seafood require a better understanding of human dietary MP exposure including extensive monitoring. While conventional techniques for MP analysis like infrared or Raman microspectroscopy provide detailed particle information, they are limited by low sample throughput, particularly when dealing with high particle numbers in seafood due to matrix-related residues. Consequently, more rapid techniques need to be developed to meet the requirements of large-scale monitoring. This study focused on semi-automated fluorescence imaging analysis after Nile red staining for rapid MP screening in seafood. By implementing RGB-based fluorescence threshold values, the need for high operator expertise to prevent misclassification was addressed. Food-relevant MP was identified with over 95% probability and differentiated from natural polymers with a 1% error rate. Comparison with laser direct infrared imaging (LDIR), a state-of-the-art method for rapid MP analysis, showed similar particle counts, indicating plausible results. However, highly variable recovery rates attributed to inhomogeneous particle spiking experiments highlight the need for future development of certified reference material including sample preparation. The proposed method demonstrated suitability of high throughput analysis for seafood samples, requiring 0.02-0.06 h/cm2 filter surface compared to 4.5-14.7 h/cm with LDIR analysis. Overall, the method holds promise as a screening tool for more accurate yet resource-intensive MP analysis methods such as spectroscopic or thermoanalytical techniques.

Spectral Investigations of Fluorescence Tracers in Automotive and Aviation Fuels under Cryogenic Conditions.

This study investigated spectral laser-induced fluorescence signals of dyes in fuels for automotive and aerospace applications under low temperatures and cryogenic conditions down to 183 K. For this purpose, a fluorescence chamber was developed based on cooling with liquid nitrogen. The design enabled a minimal inner chamber temperature of 153 K. Furthermore, the applicability of two-color LIF for liquid thermometry was evaluated under these conditions. The temperature determination was based on the temperature-sensitive fluorescence intensity ratio of the special dyes doped into the fuels determined in suitable spectral regions, which represented common bandpass filters. For this purpose, the fluorescence signals of the dye doped into the gasoline and jet fuel surrogate isooctane were tested as well as blends of isooctane and the ethanol biofuels E20 (comprising 80 vol.% isooctane and 20 vol.% ethanol), E40, and E100. Additionally, a realistic multi-component fuel Jet A-1 mixed with a suitable fluorescence dye was investigated. E100 was doped with Eosin-Y, and the remaining fuels were doped with Nile red. Temperature-dependent spectral LIF intensities were recorded in the range of 183 K-293 K, which simulate extreme environments for aerospace and automotive applications. Frozen fuel-dye mixtures cause significant extinction effects and prevent sufficient signal detection at low and cryogenic temperatures, defining the detection limit. A temperature decrease led to a spectral shift in the emission peaks of E100 doped with Eosin-Y toward shorter wavelengths, while the spectra of mixtures doped with Nile red were shifted toward longer wavelengths. The suggested bandpass filters produced the temperature-sensitive intensity ratio (the average over the temperature interval) of the dyes with the largest sensitivity for Jet A-1 (5.2%/K), followed by E100 (4.95%/K), E40 (4.07%/K), E20 (3.23%/K), and isooctane (3.07%/K), even at cryogenic temperatures.

Optimization of Cultivation Conditions of Native Microalga Scenedesmus quadricauda and Evaluation of Lipids for Enhanced Biodiesel Production.

Biofuels are considered to be among the primary alternatives to the use of fossil fuels. These fuels, made from feedstock or waste raw materials, have the advantage of being renewable and contributing much less to global warming. Microalgae are a promising biodiesel source. Microalgae, unlike traditional crops that are now used to make commercialized biodiesel, may be grown on non-agricultural land and has a greater capacity for growth and yield. Cultivation has been considered as a critical stage in the generation of biofuels. The goal of the present study is to learn that Scenedesmus quadricauda has a potential for biodiesel production in the near future. Optimization studies revealed that BG-11 medium, temperature of 25 °C, pH 7.0, glucose and sucrose (as carbon sources), static condition (for lipid accumulation) & shaking condition (for biomass yield), cultivation days of 18, 21, and 24 day, NaNO3 dosing of 1.0 mM followed by 0.8 mM (on 5th day of cultivation), 3% yeast extract dosing, 3000 lx light intensity, photoperiod cycles of 24L/0D (for biomass yield) and 18L/6D (for lipid production) and 10 mM concentration of NaCl (salinity stress) can be regarded as best suited physio-biochemical parameters for efficient biomass and lipid yield from S. quadricauda. FTIR indicated presence of various stretching of carbohydrates and lipids that again is supporting biodiesel production capability of S. quadricauda. SEM showed that cells of S. quadricauda under stress conditions became fragmented separated from coenobium and were not so compactly arranged. Present optimization studies along with Nile red fluorescence, FTIR and SEM revealed that S. quadricauda could be a suitable candidate to produce good quality biofuel and that also in stress conditions.

Sensitive and discriminative detection of cysteine by a Nile red-based NIR fluorescence probe.

Cysteine (Cys) is a significant biological mercaptan that achieves key roles in several important physiological processes, such as reversible redox homeostasis in living organisms. Abnormal levels of Cys in the human body are directly related to many diseases. In this work, we constructed a sensitive sensor (Cys-NR) by connecting a Cys recognition group to a Nile red derivative. Due to photo-induced electron transfer (PET), the Cys-NR probe showed little fluorescence at 650 nm. With the addition of Cys to the assay solution, the chlorine unit of the probe was substituted by the thiol group of Cys. Further, the amino and sulfhydryl groups in cysteine underwent an intramolecular rearrangement, which led to the Cys-NR probe water solution turning from colorless to pink with an enhancement in fluorescence. The red fluorescence at 650 nm increased about 20 times. Based on the turn-on signal, a selective Cys detection method is developed. The probe signal is not affected by various potential interferences or other competing biothiols and the limit of detection (LOD) is determined to be 0.44 µM. In addition, the probe is further employed for imaging of Cys in live cells, revealing good biological imaging ability that could provide a new way of intracellular Cys detection.

Nile red-based lipid fluorometry protocol and its use for statistical optimization of lipids in oleaginous yeasts.

As lipogenic yeasts are becoming increasingly harnessed as biofactories of oleochemicals, the availability of efficient protocols for the determination and optimization of lipid titers in these organisms is necessary. In this study, we optimized a quick, reliable, and high-throughput Nile red-based lipid fluorometry protocol adapted for oleaginous yeasts and validated it using different approaches, the most important of which is using gas chromatography coupled to flame ionization detection and mass spectrometry. This protocol was applied in the optimization of the concentrations of ammonium chloride and glycerol for attaining highest lipid titers in Rhodotorula toruloides NRRL Y-6987 and Yarrowia lipolytica W29 using response surface central composite design (CCD). Results of this optimization showed that the optimal concentration of ammonium chloride and glycerol is 4 and 123 g/L achieving a C/N ratio of 57 for R. toruloides, whereas for Y. lipolytica, concentrations are 4 and 139 g/L with a C/N ratio of 61 for Y. lipolytica. Outside the C/N of 33 to 74 and 45 to 75, respectively, for R. toruloides and Y. lipolytica, lipid productions decrease by more than 10%. The developed regression models and response surface plots show the importance of the careful selection of C/N ratio to attain maximal lipid production. KEY POINTS: • Nile red (NR)-based lipid fluorometry is efficient, rapid, cheap, high-throughput. • NR-based lipid fluorometry can be well used for large-scale experiments like DoE. • Optimal molar C/N ratio for maximum lipid production in lipogenic yeasts is ~60.

Biological responses of Chironomus sancticaroli to exposure to naturally aged PP microplastics under realistic concentrations.

Microplastic (MP) is yet another form of chronic anthropogenic contribution to the environment. MPs are plastic particles (<5 mm) that have been widely found in the most diverse natural environments, but their real impacts on ecosystems are still under investigation. Here, we studied the toxicity of naturally aged secondary polypropylene (PP) MPs after constant exposure to ultraviolet radiation (26 µm) to the third instar larvae of Chironomus sancticaroli, a dipteran species. The concentrations tested were 13.5; 67.5; and 135 items g-1 of dry sediment. C. sancticaroli organisms were investigated for fragment ingestion, mortality and changes in enzymatic biomarkers after 144 h of exposure. The organisms were able to ingest MPs from the first 48 h, and the amount of items internalized was dose-dependent and time-dependent. Overall, the results show that mortality was low, being significant at the lowest and highest concentrations (13.5 and 135 items g-1). Regarding changes in biochemical markers, after 144 h MDA and CAT activities were both significantly altered (increased and reduced, respectively), while SOD and GST levels were unchanged. In the present study, naturally aged polypropylene MPs induced biochemical toxicity in C. sancticaroli larvae, with toxicity being higher according to exposure time and particle concentration.

Combined approaches for detecting polypropylene microplastics in crop plants.

Microplastics (MPs) pollution in the terrestrial environment causes accumulation in crop plants. Consumption of these plants may have negative effects on human health. Therefore, it is crucial to analyze MPs accumulation in the plants. The aim of this study is to determine polypropylene (PP) particles in plants exposed to label-free PP for 75 days. In order to extract PP from organic matter, a two-step alkaline and wet peroxide oxidation chemical digestion method was applied to the roots, stems, and leaves of maize and wheat. The PP particles in the digested solutions were detected by the Nile red staining method, which has not been used previously in the detection of MPs in plants. Nile red stained PP particles mostly accumulated in the roots of wheat and the stems of maize plants. Statistical analysis revealed that the maize deposited more and larger PP particles regardless of the location. Moreover, the presence of PP particles in the digestion solutions was proved by the heating method. The PP particles on the glass slides were transformed into different shapes due to melting.

The optimal combination of Nile red identification, colony polymerase chain reaction, and gas chromatography detection methods in screening for polyhydroxyalkanoicate-producing bacteria.

The study devised a detection process combining Nile red-containing media, polymerase chain reaction (PCR), and gas chromatography (GC) to evaluate the possibility of microbes becoming polyhydroxyalkanoate (PHA) producers. The Nile red and PCR detection steps of designating PHA producers had true positive rates of 39.4% and 40%, respectively, and the use of GC analysis as the final step yielded accurate results for the production types and yields of PHAs. When the number of screening samples was up to 102, connecting all three inspection methods in tandem generated economic benefits. When up to 105 samples were screened, the use of all three detection methods reduced the cost to 3% of the cost and the time consumed 6% of using just Nile red plus GC or PCR plus GC. However, when the sum of samples exceeded 108, the cost of combining the three methods exceeds 1 million US dollars and was excessive; here, the combination of Nile red plus PCR could be considered, even though the true positive rate was only 30.7%.

Microplastics generation and concentration during mechanical-biological treatment of mixed municipal solid waste.

Mechanical-biological treatment (MBT) is a popular solution for the processing of mixed municipal solid waste (MSW). However, it is assumed that the treatment processes can lead to the generation of microplastics in large quantities and their concentration in the organic output. Organic outputs from MBT as a source of microplastics are still poorly understood. The current article aims to fill this gap and investigate microplastics formation during MBT and their abundance in ready stabilized organic output. Seasonal samples were taken from the four stages of the possible microplastics pathway in MBT to study changes in microplastics numerical and mass concentration, shape and size. Large microplastics were identified by Fourier transform infrared spectroscopy, and small microplastics by Nile Red dye staining method. The results showed that both mechanical pre-treatment and aerobic treatment had a significant impact on microplastics formation, while mechanical post-treatment only resulted in the enrichment of the output with microplastics. Moreover, microplastics became finer during treatment. Microplastics abundance in ready organic output ranged from 8925 ± 1344 particles/kg in winter 2021 to 17407 ± 4319 particles/kg in summer 2020, and up to 160.5 t of microplastics were emitted from the Kaunas MBT treatment facility during the study year. In addition, a relationship between the microplastics abundance and plastic content of the incoming waste was found by a regression analysis. Therefore, to reduce the formation and emission of microplastics by MBT, the organic fraction of MSW should be collected and treated separately.

In situ quantification of poly(3-hydroxybutyrate) and biomass in Cupriavidus necator by a fluorescence spectroscopic assay.

Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. KEY POINTS: • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red.

Labeling Microplastics with Fluorescent Dyes for Detection, Recovery, and Degradation Experiments.

Staining microplastics (MPs) for fluorescence detection has been widely applied in MP analyses. However, there is a lack of standardized staining procedures and conditions, with different researchers using different dye concentrations, solvents, incubation times, and staining temperatures. Moreover, with the limited types and morphologies of commercially available MPs, a simple and optimized approach to making fluorescent MPs is needed. In this study, 4 different textile dyes, along with Nile red dye for comparison, are used to stain 17 different polymers under various conditions to optimize the staining procedure. The MPs included both virgin and naturally weathered polymers with different sizes and shapes (e.g., fragments, fibers, foams, pellets, beads). We show that the strongest fluorescence intensity occurred with aqueous staining at 70 °C for 3 h with a dye concentration of 5 mg/mL, 55 mg/mL, and 2 µg/mL for iDye dyes, Rit dyes, and Nile red, respectively. Red fluorescent signals are stronger and thus preferred over green ones. The staining procedure did not significantly alter the surface, mass, and chemical characteristics of the particles, based on FTIR and stereomicroscopy. Stained MPs were spiked into freshwater, saltwater, a sediment slurry, and wastewater-activated sludge; even after several days, the recovered particles are still strongly fluoresced. The approach described herein for producing customized fluorescent MPs and quantifying MPs in laboratory-controlled experiments is both straightforward and simple.

Solubilization of Nile Red in Micelles and Protomicelles of Sodium Dodecyl Sulfate.

A spectrophotometric study of the solubilization and aggregation of the Nile red dye (NR) in premicellar and micellar aqueous solutions of sodium dodecyl sulfate (SDS) was carried out. The experiments were conducted both with saturated solutions of NR under conditions of thermodynamic equilibrium of the solution with a dye precipitate, and at a constant concentration of NR in a homogeneous solution. In the first case, it was proved theoretically and verified experimentally that with an increase in the SDS concentration, the NR concentration always increases, and at the limit of low concentrations, the dependence is linear. In both cases, the concentration of NR dimers as a function passes through a maximum in the premicellar region. There are no dimers in the micellar region. The extinction coefficients of NR monomers in SDS solutions were determined both below and above the critical micelle concentration (CMC) of SDS. A solubilization curve with branches for the premicellar and micellar regions was constructed, the intersection of which was used to find the CMC value in the system under study. The state of deep supersaturation of the NR solution in the metastable state upon dilution of the micellar system with water was studied. It was found that, in addition to dimers, molecular aggregates of higher orders were also formed.

Adaptive evolution of engineered yeast for squalene production improvement and its genome-wide analysis.

In the present study, the adaptive evolution of a metabolically engineered Saccharomyces cerevisiae strain in the presence of an enzyme inhibitor terbinafine for enhanced squalene accumulation via serial transfer leads to the development of robust strains. After adaptation for nearly 1500 h, a strain with higher squalene production efficiency was identified at a specific growth rate of 0.28 h-1 with a final squalene titer of 193 mg/L, which is 16.5-fold higher than the BY4741 and 3-fold higher over the metabolically engineered SK22 strain. Whole-genome sequencing comparison between the reference strain and the evolved variant SK23 has led to the identification of 462 single-nucleotide variants (SNVs) between both strains, with 102 SNVs affecting metabolism-related genes. It was also established that F420I mutation of ERG1 in S. cerevisiae improves squalene synthesis. Further, the effect of increased squalene on lipid droplet and neutral lipid pattern in the evolved mutant strains was investigated by fluorescent techniques proving that the neutral lipid content and clustering of lipid droplets increase with an increase in squalene.

Evaluation of ThT augmentation and RLS inner filter effect caused by highly fluorescent coumarin derivative and establishing it as true inhibitor of amyloid fibrillation.

Screening of inhibitors that slow down or suppress amyloid fibrils formation relies on some simple but sensitive spectroscopy techniques. Thioflavin T (ThT) fluorescence assay is one of the most common, amyloid specific and sensitive method. However, if an inhibitor is itself fluorescent in the ThT fluorescence range, its screening becomes complicated and require complementary assays. One of such molecules, 6, 7-dihydroxycoumarin (6, 7-DHC, also known as aesculetin, esculetin, and cichorigenin) is fluorescent in the ThT emission range and absorbs in the ThT excitation range. Therefore, it can produce a subtractive effect attributed to primary inner filter effect and/or additive effect due to its self-fluorescence in ThT assay. Our study shows that 6, 7-DHC produces an additive effect in ThT fluorescence, which is minimized at high concentration of ThT and decrease in ThT fluorescence is solely due to its inhibitory effect against HSA fibrillation. These ThT fluorescence-based results are verified through other complementary assays, such as Rayleigh and dynamic light scattering and amyloid-specific Congo red binding assay. Furthermore, hydrophobicity reduction is studied through Nile red (NR) and kinetics through far-UV circular dichroism (far-UV CD) in place of the most commonly employed ThT assay owing to extremely high fluorescence of 6, 7-DHC during initial incubation period.

The potential of fluorescent dyes-comparative study of Nile red and three derivatives for the detection of microplastics.

During the last years, microplastics in the environment came to the fore in environmental science research. For an appropriate risk assessment, it is essential to know the levels of microplastic contamination in the environment. In the field of microplastic detection, extensive research has been carried out in recent years. While common methods such as Raman spectroscopy and pyrolysis GC-MS are time-consuming and require trained staff and expensive equipment, there is the need for a cheap and easily applicable method. Staining microplastics with the fluorescent dye Nile red (NR) has a high potential to fulfill these criteria. In our work, we tested Nile red and newly developed derivatives, with the aim of achieving greater selectivity for plastic particles and more intense fluorescence. In addition, the influence of using different solvents and water at different pH values in the dyeing process was investigated by analyzing solid sample fluorescence spectra of dyed microplastics and natural particles. Finally, the method developed from the acquired knowledge was tested for sea salt. Graphical abstract.

Microplastics in lakeshore and lakebed sediments - External influences and temporal and spatial variabilities of concentrations.

Microplastics have been predominantly studied in marine environments compared to freshwater systems. However, the number of studies analyzing microplastic concentrations in water and sediment within lakes and rivers are increasing and are of utmost importance as freshwaters are major pathways for plastics to the oceans. To allow for an adequate risk assessment, detailed knowledge concerning plastic concentrations in different environmental compartments of freshwaters are necessary. Therefore, the major aim of this study was the quantification and analysis of temporal and spatial distribution of microplastics (<5 mm) in freshwater shore and bed sediments at Lake Tollense, Mecklenburg-Western Pomerania, Germany. Likewise, it addresses the hypothesis that lakes may serve as long-term storage basins for microplastics. Concentrations were investigated semi-annually over a two-year period at four sandy bank border segments representing different expositions and levels of anthropogenic influence. In addition, lakebed samples were taken along the longitudinal dimension of Lake Tollense. Mean microplastic abundances were 1,410 ± 822 particles/kg DW for lakeshore sediments and 10,476 ± 4,290 particles/kg DW for lakebed sediments. Fragments were more abundant compared to fibers in both sediment compartments. Spatial and temporal variation was especially recognized for lakeshore sediments whereas microplastic abundances in lakebed sediments did not differ significantly between sampling points and sampling campaigns. This can be related to long-term accumulation at the lakebed. Lower microplastic abundances were found within the intertidal zone at lake beaches where constant wave action reduces accumulation. Increased microplastic abundances were recognized at the beach with least anthropogenic influence but in proximity to a tributary, which may serve as microplastic input pathway into Lake Tollense due to its catchment comprising mainly agricultural areas. Furthermore, spatial variations in microplastic concentrations were related to the abundance of macroplastic items at beaches and correlated with pedologic sediment characteristics, namely the content of organic matter.

Optimization of PTFE Coating on PDMS Surfaces for Inhibition of Hydrophobic Molecule Absorption for Increased Optical Detection Sensitivity.

Polydimethylsiloxane (PDMS) is a polymer widely used for fabrication and prototyping of microfluidic chips. The porous matrix structure of PDMS allows small hydrophobic molecules including some fluorescent dyes to be readily absorbed to PDMS and results in high fluorescent background signals, thereby significantly decreasing the optical detection sensitivity. This makes it challenging to accurately detect the fluorescent signals from samples using PDMS devices. Here, we have utilized polytetrafluoroethylene (PTFE) to inhibit absorption of hydrophobic small molecules on PDMS. Nile red was used to analyze the effectiveness of the inhibition and the absorbed fluorescence intensities for 3% and 6% PTFE coating (7.7 ± 1.0 and 6.6 ± 0.2) was twofold lower compared to 1% and 2% PTFE coating results (17.2 ± 0.5 and 15.4 ± 0.5). When compared to the control (55.3 ± 1.6), it was sevenfold lower in background fluorescent intensity. Furthermore, we validated the optimized PTFE coating condition using a PDMS bioreactor capable of locally stimulating cells during culture to quantitatively analyze the lipid production using Chlamydomonas reinhardtii CC-125. Three percent PTFE coating was selected as the optimal concentration as there was no significant difference between 3% and 6% PTFE coating. Intracellular lipid contents of the cells were successfully stained with Nile Red inside the bioreactor and 3% PTFE coating successfully minimized the background fluorescence noise, allowing strong optical lipid signal to be detected within the PDMS bioreactor comparable to that of off-chip, less than 1% difference.