Nile Red Dye Research Articles

Application of fluorescent microscopy and Fourier-transform infrared spectroscopy for analysis of microplastics in Can Gio seawater

Microplastics (MPs) pollution has become a global challenge due to their persistent properties and harm to nature and human health. Observation of MPs pollution, especially in marine environments, is a critical concern in environmental analysis. In this work, we validate the method for detecting the presence of MPs in seawater by using fluorescence microscopy with Nile Red dye combined with Attenuated Total Reflectance Fourier-transform infrared spectroscopy (ATC-FTIR). The specificity and accuracy of this method were evaluated by analyzing the standard spiked samples. Five standard MPs and chitin samples used in this study were polyamide (PA), polyethylene (PE), polyethylene terephthalate (PET), poly(methyl methacrylate) (PMMA), and polyvinyl chloride (PVC) with a diameter from 5 to 300µm. The obtained recovery was 88.3-96.7%, with the related standard deviation (RSD%) in the range of 4.6-11.0%. This method was applied to determine microplastics in 27 seawater samples at three locations in Can Gio Sea, Ho Chi Minh City. The results showed the pollution level of each type of MP was significantly varied, with PE and PET being the highest, while there was almost no PMMA in seawater samples. ® 2023 Journal of Science and Technology - NTTU

Microplastic Identification and Quantification in Biological Samples

Background 
 The convenience and durability of plastics has made it a staple in the manufacturing industry, but without adequate recycling, these plastics are filling landfills at an alarming rate. This plastic waste is broken down into micro- and nano-plastics that have been found in both marine and terrestrial environments. The potential ramifications of accumulating plastic particles in the environment are of urgent attention as humans are exposed to these particles through ingestion, inhalation, and dermal exposure. Due to the lack of consensus of the effect microplastics have on the coagulation cascade, our aim was to form clots under various conditions so that a comparison can be drawn. 
 Methods 
 We utilized varying sizes (500-2,000 nm) and concentrations (25-250 µg/ml) of non-functionalized polystyrene (nPS) microplastics to characterize their effect on clot size, strength, and anatomy using ex-vivo shear flow clot formation of human whole blood via a Chandler loop apparatus. Additionally, we studied the effect of fluorescent Nile red dye (NR, ex/em: 545/625 nm) on clotting dynamics and visualization of microplastic particles via fluorescence microscopy. 
 Results 
 Ex-vivo clots were made (422 s-1 shear at increasing nanoplastic concentrations), sectioned, and utilized for quantification optimization in histological processing to preserve plastic integrity and fluorescent signal. NR-dyed microplastics were shown to produce sufficient fluorescent signal for imaging. Images were taken at various magnifications (4x-40x) and exposure times (100µs-500ms) using brightfield and fluorescent microscopy (Cy3 filter) on a Nikon Eclipse Ni microscope and a Nikon DS-Ri2 Microscope camera. 
 Potential Impacts 
 These results highlight the need for further analysis of the effects of microplastics on the coagulation cascade, as well as further optimization of techniques used to identify and quantify microplastics in complex biological samples. 

A Nile red dye cathode with an asymmetric redox unit for lithium organic batteries.

A Nile red (NR) dye cathode with an asymmetric redox structure of para CN and CO bonds was developed for use in an efficient lithium organic battery with a good capacity of 125 mA h g-1 and two visible discharge/charge voltage plateaus (≈2.0 V and ≈1.7 V). The NR cathode demonstrated the advantages of employing cost-effective dyes to achieve multigradient voltage platform regulation.

Antibacterial activity of hydrophobicity modulated cationic polymers with enzyme and pH-responsiveness.

The membrane lipid compositions of prokaryotic and eukaryotic cells are inherently different in many aspects, although some similarities exist in their structure and composition. Therefore, selective targeting of membrane lipids with a compound of therapeutic value, such as an antibacterial copolymer, is often challenging. Hence, developing an ideal copolymer with antibacterial properties demands hydrophobicity/hydrophilicity balance with a high biosafety profile. To integrate hydrophobic/hydrophilic balance and cationic charge in an alternating antibacterial copolymer with enzyme and pH-responsiveness, a lysine appended styrenic monomer was copolymerized with a fatty acid (octanoic acid (OA) or myristic acid (MA)) tethered maleimide monomer via reversible addition-fragmentation chain transfer (RAFT) polymerization. A range of microscopic analyses, including dynamic light scattering (DLS), confirmed the formation of nanoaggregates (size ∼30-40 nm) by these polymers in aqueous solution with positive zeta potential (cationic surface charge). Hydrophobic Nile red (NR) dye was successfully encapsulated in the nanoaggregates, and the in vitro release kinetics of the NR dye were monitored at different pHs and in the presence or absence of esterase/lipase. The in vitro release kinetics of NR revealed ∼85% dye release in the presence of pH 5.5 and lipase, suggesting their suitability for pH/enzyme-triggered therapeutic payload delivery. The standard broth microdilution assay showed significant bactericidal activity against both Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria with an MIC50 value <30 μg mL-1. The effect of polymeric nanoaggregates on bacterial morphology and in vitro survival was further confirmed by field emission scanning electron microscopy (FESEM), agar gel disk diffusion assay, and bacterial live/dead cell count. The significantly low hemolytic activity against red blood cells (RBCs) (HC50 >103 μg mL-1) and nontoxic effect on human intestinal epithelial cells (INT 407) (EC50 >500 μg mL-1) ensure that the polymer nanoaggregates are safe for in vivo use and can serve as a potent antibacterial polymer.

Optically monitoring the microenvironment of a hydrophobic cargo in amphiphilic nanogels: influence of network composition on loading and release.

Amphiphilic nanogels (ANGs) are promising carriers for hydrophobic cargos such as drugs, dyes, and catalysts. Loading content and release kinetics of these compounds are controlled by type and number of hydrophobic groups in the amphiphilic copolymer network. Thus, understanding the interactions between cargo and colloidal carrier is mandatory for a tailor-made and cargo-specific ANG design. To systematically explore the influence of the network composition on these interactions, we prepared a set of ANGs of different amphiphilicity and loaded these ANGs with varying concentrations of the solvatochromic dye Nile Red (NR). Here, NR acts as a hydrophobic model cargo to optically probe the polarity of its microenvironment. Analysis of the NR emission spectra as well as measurements of the fluorescence quantum yields and decay kinetics revealed a decrease in the polarity of the NR microenvironment with increasing hydrophobicity of the hydrophobic groups in the ANG network and dye-dye interactions at higher loading concentrations. At low NR concentrations, the hydrophobic cargo NR is encapsulated in the hydrophobic domains. Increasing NR concentrations resulted in probe molecules located in a more hydrophilic environment, i.e., at the nanodomain border, and favored dye-dye interactions and NR aggregation. These results correlate well with release experiments, indicating first NR release from more hydrophilic network locations. Overall, our findings demonstrate the importance to understand carrier-drug interactions for efficient loading and controlled release profiles in amphiphilic nanogels.

Photoaging enhances combined toxicity of microplastics and tetrabromobisphenol A by inducing intestinal damage and oxidative stress in Caenorhabditis elegans

Microplastics (MPs) are emerging environmental contaminants that often co-exist with tetrabromobisphenol A (TBBPA) in the environment. However, the joint effect of TBBPA and photoaged MPs at ambient concentrations remains unknown largely. In this study, the combined toxicity of ultraviolet-aged polystyrene (UV-PS) and TBBPA was investigated in Caenorhabditis elegans. UV irradiation could change the physical and chemical characteristics of polystyrene (PS), and UV-PS (90.218 μg/g) showed a stronger adsorption capacity than PS of 79.424 μg/g. Toxicity testing showed that 1 μg/L UV-PS enhanced the toxic effect of 1 μg/L TBBPA by reducing body length, locomotion behavior, and brood size in nematodes. Using ROS production, lipofuscin accumulation, and expression of gst-4::GFP as endpoints, the combined exposure of UV-PS and TBBPA induced stronger oxidative stress than TBBPA alone. Joint exposure to UV-PS and TBBPA significantly increased of Nile red and blue food dye in its intestinal tract compared to that in the TBBPA exposure group, indicating that co-exposure enhanced intestinal permeability. After co-exposure to UV-PS and TBBPA, the expression of the associated genes detected increased significantly. Therefore, UV-PS enhances the adverse effects of TBBPA through intestinal damage and oxidative stress in nematodes. These findings suggest that the co-presence of photoaged PS and TBBPA results in high environmental risks.

Application of fluorescent microscopy and Fourier-transform infrared spectroscopy for analysis of microplastics in Can Gio seawater

Microplastics (MPs) pollution has become a global challenge due to their persistentproperties and harm to nature and human health. Observation of MPs pollution,especially in marine environments, is a critical concern in environmental analysis. Inthis work, we validate the method for detecting the presence of MPs in seawater byusing fluorescence microscopy with Nile Red dye combined with Attenuated TotalReflectance Fourier-transform infrared spectroscopy (ATC-FTIR). The specificity andaccuracy of this method were evaluated by analyzing the standard spiked samples.Five standard MPs and chitin samples used in this study were polyamide (PA),polyethylene (PE), polyethylene terephthalate (PET), poly(methyl methacrylate)(PMMA), and polyvinyl chloride (PVC) with a diameter from 5 to 300μm. Theobtained recovery was 88.3-96.7%, with the related standard deviation (RSD%) in therange of 4.6-11.0%. This method was applied to determine microplastics in 27seawater samples at three locations in Can Gio Sea, Ho Chi Minh City. The resultsshowed the pollution level of each type of MP was significantly varied, with PE andPET being the highest, while there was almost no PMMA in seawater samples.® 2023 Journal of Science and Technology - NTTU

Systematic analysis of proximal midgut- and anorectal-originating contractions in larval zebrafish using event feature detection and supervised machine learning algorithms.

Zebrafish larvae are translucent, allowing in vivo analysis of gut development and physiology, including gut motility. While recent progress has been made in measuring gut motility in larvae, challenges remain which can influence results, such as how data are interpreted, opportunities for technical user error, and inconsistencies in methods. To overcome these challenges, we noninvasively introduced Nile Red fluorescent dye to fill the intraluminal gut space in zebrafish larvae and collected serial confocal microscopic images of gut fluorescence. We automated the detection of fluorescent-contrasted contraction events against the median-subtracted signal and compared it to manually annotated gut contraction events across anatomically defined gut regions. Supervised machine learning (multiple logistic regression) was then used to discriminate between true contraction events and noise. To demonstrate, we analyzed motility in larvae under control and reserpine-treated conditions. We also used automated event detection analysis to compare unfed and fed larvae. Automated analysis retained event features for proximal midgut-originating retrograde and anterograde contractions and anorectal-originating retrograde contractions. While manual annotation showed reserpine disrupted gut motility, machine learning only achieved equivalent contraction discrimination in controls and failed to accurately identify contractions after reserpine due to insufficient intraluminal fluorescence. Automated analysis also showed feeding had no effect on the frequency of anorectal-originating contractions. Automated event detection analysis rapidly and accurately annotated contraction events, including the previously neglected phenomenon of anorectal contractions. However, challenges remain to discriminate contraction events based on intraluminal fluorescence under treatment conditions that disrupt functional motility.

Bioplastic (Polyhydroxyalkanoate) producing Indigenous Bacteria from Agricultural Soil Samples

Polyhydroxyalkanoate (PHA) is the naturally occurring biopolymer produced by the microorganisms. It is biodegradable and having physicochemical properties similar to conventional synthetic plastics and it can be used as an alternative to plastics. PHA is produced by microorganisms including bacteria, archaea and fungi. It gets accumulated inside the microorganisms as crystal in the presence of excess source of carbon, nitrogen, sulphur, oxygen and limited source of other nutrients. In this study PHA producing strain was isolated from agricultural soil sample. All the isolates obtained were screened for PHA production by using Nile red and Sudan black dyes. The potential isolate was characterized by biochemical tests and identified using 16S rRNA sequencing. The isolate showed sequence similarity with Priestia aryabhattai B8W22 and was designated as Priestia aryabhattai VITJK01. For PHA production, seed culture was prepared and inoculated in mineral salt medium containing glucose as a carbon source. PHA was extracted from cell dry weight biomass using methanolysis. It produced polyhydroxybutyrate (PHB) compounds including dodecane, penta decanoic acid, octadecanoic acid and heptadecanoic acid identified by Gas Chromatography Mass Spectrometry. Based on the results, it can be concluded that Priestia aryabhattai VITJK01 is a potential source for PHA/PHB production.

A non-fluorinated superhydrophobic PDMS/STA/TiO2 coating with photocatalysis and environmental stability properties by one-step cold spraying

Constructing a titanium dioxide (TiO2) superhydrophobic surface that is both environmentally friendly and long-term stable, while also possessing photocatalytic properties, is a considerable challenge. In this work, instead of a perfluorinated modifier, we used stearic acid (STA) to modify TiO2 nanoparticles. The particles were further utilized to facilitate the rough structure of superhydrophobic surfaces, making these surfaces fluoride-free and environmentally friendly in practical applications. STA/TiO2 nanoparticles and Polydimethylsiloxane (PDMS) were mixed in an ethyl acetate solution to form the PDMS/STA/TiO2 emulsion, which was used to prepare the PDMS/STA/TiO2 coating by a one-step cold spraying. PDMS was introduced into the coating, providing the lower surface energy, adhesiveness, and protection from photo-oxidative damage. It is found that the non-fluorinated PDMS/STA/TiO2 coating exhibits good superhydrophobicity with a water contact angle of 159.7° and a sliding angle of 2.9°. Under ultraviolet (UV) irradiation, the coating displayed excellent photocatalytic degradation activity towards Methylene Blue, Methyl Orange, and Nile Red dyes. Moreover, the coating can withstand sandpaper abrasion, high temperatures and exposure to droplets of different pH values, indicating good chemical, mechanical and thermal stability. The coating presented strong durability, maintaining its superhydrophobicity even after being exposed to air for 3 months or UV irradiation for 5 h. Besides, the coating demonstrates good self-cleaning performance and adaptability. The large-area superhydrophobic surface made by simple and convenient one-step cold spraying is expected to have practical applications in shipbuilding, construction, textiles, and other industries.

Synthesis and self-assembling of hyaluronan grafted with ceramide NP for topical drug delivery

In this work, amphiphilic hyaluronan was synthesized by grafting succinylated N-oleoyl-phytosphingosine via esters bonds. Succinylated N-oleoyl-phytosphingosine (sCER) was first prepared by esterification of hydroxyl moieties of the ceramide with succinic anhydride. The esterification of hyaluronan was governed by crowding effect. The oligomeric HA-sCER derivatives exhibited a strong self-aggregation as evidenced by a very low critical aggregation concentration (1.9 μg mL−1), higher pyrene binding constant (KB), and the smallest particle size (30 nm) in solution. The self-aggregation properties demonstrated to be a function of the substitution degree and molecular weight of HA. The prepared derivatives were non-cytotoxic towards cell lines NIH-3T3. Nanoparticles prepared using oligomeric HA-sCER derivatives improved the penetration of Nile red dye through the stratum corneum due to their smaller size (≤50 nm). The fluorescence intensity localized at the stratum corneum was higher for oligomeric HA-sCER. A significant inhibition of the pro-inflammatory cytokine interleukin-6 production was observed in vitro in macrophages differentiated from THP-1 cells. These findings showed that HA-sCER constituted a promising active ingredient for cosmetics use.

Spatial distribution of atmospheric microplastics in bulk-deposition of urban and rural environments – A one-year follow-up study in northern Germany

Atmospheric microplastic deposition rates play a crucial role for calculating the input of microplastics in the environment and to further understand pollution patterns. In this study, the spatial and temporal distribution of atmospheric microplastic particles in urban and rural areas of Northern Germany was investigated. Therefore, eleven structurally diverse locations in Hamburg and Mecklenburg-Western Pomerania were equipped with bulk-deposition samplers in triplicates and sampled monthly between August 2019 and July 2020. The resulting 306 samples were treated with hydrogen peroxide (30 %) and sodium hypochlorite (6–14 %) to digest biological organic matter. The filters were subsequently stained with the lipophilic dye Nile Red and underwent visual microplastic identification via fluorescence microscopy. Fragments and fibers were quantified down to a cut-off size of 10 μm. The polymer composition of microplastic particles was investigated along a subset of particles via μ-Raman spectroscopy.The microplastic deposition rate for Northern Germany (89 ± 61 MP/m2/day) is in the same order of magnitude as those reported by previous studies. Significant differences in microplastic deposition rates were found between urban and rural sampling sites. Population density was identified as an important factor for greater amounts of microplastics and higher shares of fibers in urban samples. Special attention was given to the canopy cover at two forested sampling sites and an influence of the comb-out effect on atmospheric microplastic deposition was detected.

Synthesis of Multifunctional Polymersomes Prepared by Polymerization-Induced Self-Assembly.

Polymersomes are an exciting modality for drug delivery due to their structural similarity to biological cells and their ability to encapsulate both hydrophilic and hydrophobic drugs. In this regard, the current work aimed to develop multifunctional polymersomes, integrating dye (with hydrophobic Nile red and hydrophilic sulfo-cyanine5-NHS ester as model drugs) encapsulation, stimulus responsiveness, and surface-ligand modifications. Polymersomes constituting poly(N-2-hydroxypropylmethacrylamide)-b-poly(N-(2-(methylthio)ethyl)acrylamide) (PHPMAm-b-PMTEAM) are prepared by aqueous dispersion RAFT-mediated polymerization-induced self-assembly (PISA). The hydrophilic block lengths have an effect on the obtained morphologies, with short chain P(HPMAm)16 affording spheres and long chain P(HPMAm)43 yielding vesicles. This further induces different responses to H2O2, with spheres fragmenting and vesicles aggregating. Folic acid (FA) is successfully conjugated to the P(HPMAm)43, which self-assembles into FA-functionalized P(HPMAm)43-b-P(MTEAM)300 polymersomes. The FA-functionalized P(HPMAm)43-b-P(MTEAM)300 polymersomes entrap both hydrophobic Nile red (NR) and hydrophilic Cy5 dye. The NR-loaded FA-linked polymersomes exhibit a controlled release of the encapsulated NR dye when exposed to 10 mM H2O2. All the polymersomes formed are stable in human plasma and well-tolerated in MCF-7 breast cancer cells. These preliminary results demonstrate that, with simple and scalable chemistry, PISA offers access to different shapes and opens up the possibility of the one-pot synthesis of multicompartmental and responsive polymersomes.

In Vivo Laser-Induced Vasoactive Microenvironmental Setting via a Stimuli-Responsive Microstructured Depot.

A stimuli-responsive polymeric three-dimensional microstructured film (PTMF) is a 3D structure with an array of sealed chambers on its external surface. In this work, we demonstrate the use of PTMF as a laser-triggered stimulus-response system for local in vivo targeted blood vessels stimulation by vasoactive substances. The native vascular networks of the mouse mesentery were used as model tissues. Epinephrine and KCl were used as vasoactive agents that were sealed into individual chambers upon precipitation in the amount of pictograms. We demonstrated the method for non-damaged one-by-one chamber activation using a focused 532 nm laser light passed through biological tissues. To avoid laser-induced photothermal damage to biological tissues, the PTMF was functionalized with Nile Red dye, which effectively absorbs laser light. Chemically stimulated blood vessel fluctuations were analyzed using digital image processing methods. Hemodynamics changes were measured and visualized using the particle image velocimetry approach.

Genetic Targeting of Solvatochromic Dyes for Probing Nanoscale Environments of Proteins in Organelles.

A variety of protein tags are available for genetically encoded protein labeling, which allow their precise localization and tracking inside the cells. A new dimension in protein imaging can be offered by combining protein tags with polarity-sensitive fluorescent probes, which provide information about local nanoscale environments of target proteins within the subcellular compartments (organelles). Here, we designed three fluorescent probes based on solvatochromic nile red dye, conjugated to a HaloTag reactive targeting group through polyethylene glycol linkers of varying lengths. The probe with medium linker length, NR12-Halo, was found to label specifically a large variety of proteins localized in defined cell compartments, such as plasma membranes (outer and inner leaflets), endoplasmic reticulum, Golgi apparatus, cytosol, microtubules, actin, and chromatin. Owing to its polarity-sensitive fluorophore, the probe clearly distinguished the proteins localized within apolar lipid membranes from other proteins. Moreover, it revealed dramatic changes in the environment during the life cycle of proteins from biosynthesis to their expected localization and, finally, to recycling inside lysosomes. Heterogeneity in the local polarity of some membrane proteins also suggested a formation of low-polar protein aggregates, for example, within cell-cell contacts. The approach also showed that mechanical stress (cell shrinking by osmotic shock) induced a general polarity decrease in membrane proteins, probably due to the condensation of biomolecules. Finally, the nanoenvironment of some membrane proteins was affected by a polyunsaturated fatty acid diet, which provided the bridge between organization of lipids and proteins. The developed solvatochromic HaloTag probe constitutes a promising tool for probing nanoscale environments of proteins and their interactions within subcellular structures.

148 Development of a test Method to Determine Inhalation Exposure of Microplastic Particles when Reusable Disposable Masks in Place

Abstract Situation/broad aims The SARS-CoV-2 pandemic has swept the globe since late 2019 and has driven explosive growth in the use of masks that has arisen an emerging concern of inhaled microplastic particles (MPs) when reuse disposable masks. In this study, we proposed a new methodology to determine whether the inhaled particles of microplastics emitted from the disposable masks when reusable mask in place as source control. Methods All experiments were conducted using a head-form manikin with a constant inspiratory flow at a simulated chamber. One commercially available ASTM Level 1 face (or surgical) mask with three-layer material, namely polypropylene (outer and middle layers), and polyethylene (inner layer) was used as test specimen to characterize the inhalation exposure of MPs. Brand new and reusable masks with and without a hand rubbing were compared. Particle physical characterization and morphology were determined using fluorescent staining with Nile red dye and scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) as well. The multiple-path particle dosimetry model was utilized to calculate the particle deposition in the respiratory tracts of human adults for emitted MPs fallen within the micrometer size range. Outcomes of the work: In this study, we proposed a method that the incorporation of scanning electron microscopy and infrared spectroscopy into the current fluorescent staining method to enhance the detection of MPs for qualitative analysis, may be more predictive of fragment microplastics are sourced from the different layer of disposable mask with a hand rubbing when in use.

Facile Suzuki Coupling Strategy toward New Nile Red Derivatives with Improved Two‐Photon Brightness

AbstractChemical fine‐tuning of fluorophores is a pivotal step towards development of next generation fluorescent dyes for microscopy. With the advent of high‐resolution two‐photon excitation fluorescence imaging, there is a growing demand for very sensitive laser dyes that can be efficiently excited using commercial Ti:sapphire laser sources in the first near‐infrared window (NIR‐I, 780–1020 nm). Using the fluorescent dye Nile Red as the lead structure, we report a robust and concise Suzuki coupling approach for the synthesis of 14 new Nile Red analogues that feature extended π ring systems and diverse functionalities. For this set, we gauged their two‐photon excitation efficiency in NIR‐I as well as evaluated their general fluorescent properties (emission wavelength, Stokes shift, quantum yield and solvatochromism). Several of the new fluorophores were found to display very favorable characteristics. In particular, the derivative featuring a 4‐aminophenyl group in the 2‐position of Nile Red exhibited extreme solvent sensitivity, and the thien‐2‐yl substituted Nile Red derivative showed significantly redshifted emission, large Stokes shift and high two‐photon brightness.

Identifying lipid particle sub-types in live Caenorhabditis elegans with two-photon fluorescence lifetime imaging.

Fat metabolism is an important modifier of aging and longevity in Caenorhabditis elegans. Given the anatomy and hermaphroditic nature of C. elegans, a major challenge is to distinguish fats that serve the energetic needs of the parent from those that are allocated to the progeny. Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy has revealed that the composition and dynamics of lipid particles are heterogeneous both within and between different tissues of this organism. Using BCARS, we have previously succeeded in distinguishing lipid-rich particles that serve as energetic reservoirs of the parent from those that are destined for the progeny. While BCARS microscopy produces high-resolution images with very high information content, it is not yet a widely available platform. Here we report a new approach combining the lipophilic vital dye Nile Red and two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for the in vivo discrimination of lipid particle sub-types. While it is widely accepted that Nile Red staining yields unreliable results for detecting lipid structures in live C. elegans due to strong interference of autofluorescence and non-specific staining signals, our results show that simple FLIM phasor analysis can effectively separate those signals and is capable of differentiating the non-polar lipid-dominant (lipid-storage), polar lipid-dominant (yolk lipoprotein) particles, and the intermediates that have been observed using BCARS microscopy. An advantage of this approach is that images can be acquired using common, commercially available 2p-FLIM systems within about 10% of the time required to generate a BCARS image. Our work provides a novel, broadly accessible approach for analyzing lipid-containing structures in a complex, live whole organism context.

Evaluation of polyhydroxyalkanoate (PHA) synthesis by Pichia sp. TSLS24 yeast isolated in Vietnam

Following the rising concern on environmental issues caused by conventional fossil-based plastics and depleting crude oil resources, polyhydroxyalkanoates (PHAs) are of great interest by scientists and biodegradable polymer market due to their outstanding properties which include high biodegradability in various conditions and processing flexibility. Many polyhydroxyalkanoate-synthesizing microorganisms, including normal and halophilic bacteria, as well as algae, have been investigated for their performance in polyhydroxyalkanoate production. However, to the best of our knowledge, there is still limited studies on PHAs-producing marine yeast. In the present study, a halophilic yeast strain isolated from Spratly Island in Vietnam were investigated for its potential in polyhydroxyalkanoate biosynthesis by growing the yeast in Zobell marine agar medium (ZMA) containing Nile red dye. The strain was identified by 26S rDNA analysis as Pichia kudriavzevii TSLS24 and registered at Genbank database under code OL757724. The amount of polyhydroxyalkanoates synthesized was quantified by measuring the intracellular materials (predicted as poly(3-hydroxybutyrate) -PHB) by gravimetric method and subsequently confirmed by Fourier transform infrared (FTIR) spectroscopic and nuclear magnetic resonance (NMR) spectroscopic analyses. Under optimal growth conditions of 35 °C and pH 7 with supplementation of glucose and yeast extract at 20 and 10 gL−1, the isolated strain achieved poly(3-hydroxybutyrate) content and concentration of 43.4% and 1.8 gL−1 after 7 days of cultivation. The poly(3-hydroxybutyrate) produced demonstrated excellent biodegradability with degradation rate of 28% after 28 days of incubation in sea water.

Analysis of selective fluorescence for the characterization of microplastic fibers: Use of a Nile Red-based analytical method to compare between natural and synthetic fibers.

The scientific community has been focusing on studying and understanding the extent of damage caused by microplastics (MPs) to flora, fauna, and humans, including the environmental and health risks associated with them. MPs with different morphologies have been described in different environments, with fibers being the most common type regardless of the environment. Various methods have been used to analyze MPs. Analytical methodologies such as visual inspection, spectroscopic methods, and others currently used to study MPs are time-consuming, and only subjective results are obtained when these methods are used for sample analysis. Researchers have used various dyes, such as Nile Red (NR), a selective fluorescent stain, to differentiate the polymers from the other sample components and address these problems. Using such dyes helps distinguish polymer particles from other contaminants present in the samples. We aimed to study the analytical process, morphology, and wettability of synthetic (such as polyethylene and polypropylene) and natural (such as linen and cotton) fibers using NR to characterize the fibers. The fibers were fragmented manually, and the samples were prepared using a cryomicrotome. The prepared samples were subjected to different NR incubation times of 30min, 24h, and 168h, and characterized under ultraviolet light using optical microscopy. We investigated the effect of NR on different fibers, and the samples selection using the fluorescence properties generated when the fibers adsorbed the NR dye. The wettabilities of the samples indicated that polyethylene and polypropylene were hydrophobic, while linen and cotton were hydrophilic. Both synthetic and natural fibers exhibited fluorescence properties in the presence of NR. This increased the complexity of executing the MP characterization process, indicating that combined methodologies and optical and chemical identification processes should be used to characterize plastic specimens efficiently. We summarize and discuss the results and findings and provide recommendations for future laboratory research on microplastic fibers focusing on (I) microplastic selection, (II) stain preparation, and (III) microplastic characterization.