Triton X-100 Research Articles

The effect of Triton X100 and Tween 80 surfactants on diesel removal from soil using electrokinetic remediation

The effect of Triton X100 and Tween 80 surfactants on diesel removal from soil using electrokinetic remediation

Characterization of a thermostable protease from Bacillus subtilis BSP strain

This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca2+ and Fe2+ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Synthesis of copolymer of aniline-o-phenylenediamine using nonionic surfactant Triton X-100: analysis of electrical conductivities and supercapacitor properties

Synthesis of copolymer of aniline-o-phenylenediamine using nonionic surfactant Triton X-100: analysis of electrical conductivities and supercapacitor properties

Synthesis of copolymer of aniline-o-phenylenediamine using nonionic surfactant Triton X-100: analysis of electrical conductivities and supercapacitor properties

Synthesis of copolymer of aniline-o-phenylenediamine using nonionic surfactant Triton X-100: analysis of electrical conductivities and supercapacitor properties

Synthesis and characterization of new 4-amino-5-methyl-4H-1,2,4-triazole-3-thiol (MMTP) Schiff base for spectrophotometric detection of iron(III) and copper(II) ions in laboratory and different water samples: A biological and molecular docking exploration

New spectrophotometric techniques were introduced for the determination of Fe(III) and Cu(II) in many different analyzed samples. The concentrations of Fe(III) and Cu(II) ions were measured in the existence of a novel Schiff base (Z)-6-(3-mercapto-5-methyl-4H-1,2,4-triazol-4-ylimino)-2-thioxotetrahydropyrimidin-4(1H)-one (MMTP). The new chelates were formed rapidly at the room temperature at 510 and 550 nm in presence of triton X-100 and CPB surfactants for Fe(III) and Cu(II), respectively. Linear calibrated graphs were obtained at concentration ranges of 1.0–130 µg mL-1 for Fe(III) and 0.5–90 µg mL-1 for Cu(II). LOD were calculated to be 1.05, 1.98 µg mL-1 and LOQ were 3.18 and 6.02 µg mL-1 for Fe(III) and Cu(II), respectively. A large number of foreign ions were not interfering in the present method. The stoichiometric composition of the chelates was studied by molar ratio and job´s methods to be found 1:2 (metal ion: ligand) ratio. The Fe(III) and Cu(II) solid chelates were prepared and characterized using IR, UV–Vis., 1HNMR , 13CNMR TG-DTG, and XRD spectroscopy. The IR spectral revealed that MMTP chelated as a bidentate ligand via NO atoms. Thermal analysis revealed the decomposition mechanism of MMTP and its chelates. A suggested octahedral geometry encompasses all of the chelates. The UV–visible spectra and magnetic moment analyses confirm the octahedral geometrical geometries. The band gap (Eg) values are 1.53 and 1.56 for Fe(III) and Cu(II) respectively. The smallest band gaps imply that these chelates are semiconductors belonging to solar materials. The in-vitro antioxidant activity of the individual chelates was evaluated using the DPPH radical scavenging assay. All compounds showed antioxidant activity with IC50 values of 0.316, 0.186 and 0.124 mg/mL for MMTP, Fe(III) and Cu(II), respectively. MMTP and its chelates were tested for their antimicrobial activity against various types of bacteria and fungi using the disc diffusion method. The Cu(II) complex showed the greatest biological activity.

Impact of surfactants used in oil and gas extraction on produced water treatment by membrane distillation

Produced water from unconventional oil and gas reservoirs often contains non-ionic surfactants that are added to the hydraulic fracturing fluid to facilitate water release and enhance gas production. This study investigates the impact of currently used surfactants (i.e., nonylphenol ethoxylates (NPEOs) and octylphenol ethoxylates (OPEOs)) and proposed alternative surfactants on the performance of membrane distillation (MD) for produced water treatment. The impact of NPEOs and OPEOs on wetting of PTFE membranes was studied as a function of their concentrations and ethoxylate (EO) chain length in a lab-scale DCMD system. Additionally, alternative surfactants, including linear alcohol ethoxylates (LAEs), branched secondary alcohol ethoxylates (BAEs), and alkyl polyglycosides (APGs), were evaluated for their potential to replace NPEOs and OPEOs. Experimental results indicate that the increase in the number of EO units in NPEOs decreased their tendency to cause membrane wetting by reducing their interaction with hydrophobic surfaces. It was also observed that LAEs and APGs that are environmentally friendly alternatives did not cause membrane wetting. This study provides valuable insights into the underlying mechanisms of surfactant-membrane interactions and offers guidelines for surfactant alternatives that can potentially improve hydraulic fracturing and lower environmental concerns while facilitating the use of MD for produced water treatment and recovery.

Spatiotemporal Filter Velocimetry With Arbitrary-Shaped Measurement Region For Accurate Measurement In The Vicinity Of A Boundary

Viscous stress at a boundary or an interface is a source of drag acting on a body or a fluid particle and therefore its measurement is of great importance for understanding the motion of the body or the fluid particle. In this study, spatiotemporal filter velocimetry (SFV: Hosokawa & Tomiyama, 2012) is extended to arbitrary quadrilateral measurement areas fitted to an interface shape for accurate measurement of flow about a deformed drop, and it is applied to flows about single drops of glycerol-water solution falling in stagnant silicon oil under clean and contaminated conditions. Figure 1 shows the trajectories of tracer particles and the measured velocity distributions. No separation is observed for the spherical and ellipsoidal drops. On the other hand, the stagnant region is observed in the dimple region of the tail of the cap drop. These results clearly indicate that the flow about the deformed drop is successfully measured by SFV with arbitrary quadrilateral measurement areas. Figure 2 shows the trajectories of tracer particles and the measured velocity distributions of single ellipsoidal drops in clean and contaminated systems. Triton X-100 was used as the surfactant and its concentration C in the drop ranges from 0.0 to 0.1 mol/m3 . The drop tail becomes flat and then slightly dimpled shape, and the internal circulation becomes weak as C increases. The internal circulation remains even at the high surfactant concentration condition under which the internal circulation in single spherical drop is fully damped (Hosokawa et al., 2017). We also evaluate interfacial velocity, interfacial shear stresses, the Marangoni stress and surfactant concentration at the interface from measured velocity distributions in the vicinity of the interface. As a result, we confirmed that SFV enables us to measure velocity distribution about a single deformed drop and enables us to evaluate interfacial velocity, interfacial shear stresses, the Marangoni stress and surfactant concentration at the interface.

Investigation of europium(III) uptake from highly sulphate solution via cloud point extraction by mono-Schiff base combined with Triton X-100

ABSTRACT Owing to its unique physico-chemical properties, europium is one of the most precious and sought-after rare earth elements in the field of high technology. The major economic and commercial importance of such an element, combined with the pollution risks associated with its intensive use, require the development of efficient and eco-compatible recovery and recycling processes. This study focuses on the recovery of europium from highly saline sulphate media (0.5 mol/L) using an environmentally friendly two-phase aqueous extraction technique (known as cloud point extraction (CPE)), using 2((phenylimino)methyl)phenol mono-Schiff base (HPIMP) as the extractant and Triton X-100 as the non-ionic surfactant. The influence of key experimental parameters such as pH, extractant concentration, surfactant concentration and separation temperature on the europium extraction process was systematically studied and optimized. Under optimum experimental conditions, a quasi-quantitative extraction with a minimal volume fraction of surfactant-rich phase (φs = 0.025), and concentration factor of (CF = 38) was achieved at pH 9.8, in one stage. The analysis of the extraction data revealed that the CPE of europium(III) takes place by a cation exchange-solvation mechanism. The stoichiometry of the complex extracted into the surfactant-rich phase was ascertained to have a composition of 1:2 [Eu:HPIMP] with the slope analysis method. A higher extraction constant was obtained for CPE compared with conventional solvent extraction, confirming the feasibility and usefulness of CPE for Eu(III) recovery. On the other hand, this new HPIMP/Triton X-100 chelating system showed superior extractability for Eu(III) in the CPE process relative to other systems reported previously.

Quantification of arsenic in real samples using a spectrophotometric cloud point extraction of the formed ion pair with astrazon orange G

A novel cloud-point extraction (CPE) procedure for the determination of ultra-trace amounts of arsenic species in real samples, purchased from the local market by spectrophotometer was developed. Inorganic arsenic species analysis in water, beverages, and foods has become increasingly important in recent years, as arsenic species are considered carcinogenic and are assessed at significant levels in samples. The technique is established on a selective ternary complex of As(V) with astrazon orange G (AOG+) in the presence of tartaric acid and polyethylene glycol tertoctylphenyl ether (Triton X-114) at pH 4.0. The calibration curve developed within range 3.0–160 ng/mL with a correlation coefficient of 0.9988 for As(V) provided a preconcentration factor of 200 and a limit of detection (3S blank/m) of 0.88 ng/mL under optimum investigation conditions. The results of molar absorptivity and Sandell sensitivity are calculated and found to be 4.38 × 105 L/mol cm and 0.018 ng cm−2, respectively. The statistical treatment of data obtained from the proposed and GF-AAS procedures are compared in terms of Student’s t-tests and variance ratio F-tests has revealed no significant differences. The methodology has been effectively confirmed by assessing real samples and comparing it to the GF-AAS method statistically.

Quantification of arsenic in real samples using a spectrophotometric cloud point extraction of the formed ion pair with astrazon orange G

A novel cloud-point extraction (CPE) procedure for the determination of ultra-trace amounts of arsenic species in real samples, purchased from the local market by spectrophotometer was developed. Inorganic arsenic species analysis in water, beverages, and foods has become increasingly important in recent years, as arsenic species are considered carcinogenic and are assessed at significant levels in samples. The technique is established on a selective ternary complex of As(V) with astrazon orange G (AOG+) in the presence of tartaric acid and polyethylene glycol tertoctylphenyl ether (Triton X-114) at pH 4.0. The calibration curve developed within range 3.0–160 ng/mL with a correlation coefficient of 0.9988 for As(V) provided a preconcentration factor of 200 and a limit of detection (3S blank/m) of 0.88 ng/mL under optimum investigation conditions. The results of molar absorptivity and Sandell sensitivity are calculated and found to be 4.38 × 105 L/mol cm and 0.018 ng cm−2, respectively. The statistical treatment of data obtained from the proposed and GF-AAS procedures are compared in terms of Student’s t-tests and variance ratio F-tests has revealed no significant differences. The methodology has been effectively confirmed by assessing real samples and comparing it to the GF-AAS method statistically.

Design, Synthesis of 3-(Aryl)-1-(2-p-tolylthiazol-4-yl)prop-2-en-1-ones as Alpha(α)-Amylase Inhibitors.

α-Amylase inhibition is vital in controlling diabetic complications. Herein, we have synthesized a hybrid scaffold based on thiazole-chalcone to access α-amylase inhbition. The proposed structures were verified with spectroscopic techniques (UV-vis, FT-IR, 1H-, 13C-NMR, and elemental analysis). The synthesized compounds were evaluated for their α-amylase and antioxidant potential. In vitro hemolytic assay was performed to test biocompatibility of all compounds. Among tested compounds, 4c (IC50= 3.8 µM), 4g (IC50= 14.5 µM), and 4f (IC50= 17.1 µM) were found excellent α-amylase inhibitors. However, none of the tested compounds exhibited significant antioxidant activity. All compounds showed less lysis than Triton X-100, but compounds 4f and 4h had the least lysis at all tested concentrations and were found to be safe for human erythrocytes. Molecular docking study was performed to evaluate the binding interactions of ligands with human pancreatic α-amylase (HPA). The binding score -8.09 to -8.507 kcal/mol revealed strong binding interactions in the ligand-protein complex. The docking results supplemented the observed α-amylase inhibition and hence augment the scaffold to serve as leads for the antidiabetic drug development.

Modulation of Triton X-100 Aqueous Micelle Interface by Ionic Liquid: A Molecular Level Interaction Studied by Time-resolved Fluorescence Spectroscopy

Background: Self-assembly structure is an important area of research for understanding biological systems, owing to its resemblance to the membrane structure of the phospholipid bilayer. In a self-assembly medium, chemical reactions and chemical or physical processes are dramatically different than the bulk phase. Understanding this process in synthesizing self-assembly structures may allow us to explore various biological processes occurring in cell membranes. Objective: The study aimed to understand water dynamics in the TX-100 micellar interface via steady state and a time-resolved fluorescence spectroscopy study. The objective was also to determine the two different ionic liquids (ILs), namely 1-butyl-3-methyl imidazolium tetrafluoroborate ([bmim][BF4]) and 1-decyl-3-methyl imidazolium tetrafluoroborate ([dmim][BF4]), inducing surfactant aggregation changes at the molecular level. Also, the focus was on determining the hydration and its dynamics at the palisade layer of TX-100 micelle in the presence of two different ionic liquids. Methods: Steady state and time-resolved fluorescence spectroscopy have been used to study TX-100 micellar systems. Employing time-resolved spectroscopy, two chemical dynamic processes, solvation dynamics and rotational relaxation dynamics, have been studied to investigate structural changes in TX100 by adding ILs. Solvation dynamics was studied by measuring the time-dependent Stokes shift of the fluorescent probe. From the Stokes shift, time-resolved emission spectra were constructed to quantify the solvation dynamics. Also, using the polarization properties of light, time-resolved anisotropy was constructed to explore the rotation relaxation of the probe molecule. Results: The absorption and emission spectra of C-153 in TX-100 were red-shifted in the presence of both the ILs. Also, the C-153 experienced faster solvation dynamics and rotational relaxation with the addition of both ILs. In our previous study, we observed a significantly increased rate of solvation dynamics with the addition of [bmim][BF4] (J. Phys. Chem. B, 115, 6957-6963) [38]. However, with the addition of the same amount of [dmim][BF4], the IL rate of solvation enhancement was more pronounced than with [bmim][BF4]. The faster solvation and rotational relaxation have been found to be associated with the penetration of more free water at the TX100 micellar stern layer, leading to increased fluidity of the micellar interface. Conclusion: Upon incorporating ILs in TX100 micelle, substantially faster solvation dynamics of water as well as rotational relaxation dynamics of C-153 have been observed. By decreasing surfactant aggregations, [bmim][BF4] ILs facilitated more water molecules approaching the TX-100 micellar phase. On the other hand, [dmim][BF4] ILs comprising mixed micelles induced even more free water molecules at the palisade layer, yielding faster solvation dynamics in comparison to pure TX-100 micelle or TX100 micelle + [bmim][BF4] ILs systems. Time-resolved anisotropy study has also supported the finding and strengthened the solvation dynamics observation.

Characterization and Animal Skin Irritations Investigation of Vemurafenib Microemulsion-Based Hydrogel Using Oily Ionic Liquid

Cutaneous melanoma accounts for a yearly mortality rate of 55,500 persons. The use of oral small-molecule kinase inhibitors, specifically targeting BRAFv600, has been licensed as the principal treatment strategy for managing both locally progressed and metastatic presentations of the condition. Approximately 30% of people on vemurafenib, a BRAFv600 inhibitor, have side effects when are taken orally. Objective: This research was attempted to develop a vemurafenib microemulsion by substituting conventional oil phases with ionic liquids (ILs). The microemulsions were created by dissolving vemurafenib in a combination of ionic liquid (1-Butyl-3-methylimidazolium hexafluorophosphate and 1-Octyl-3-methylimidazolium hexafluorophosphate) and surfactant (Triton x-100). Multiple tests were conducted to measure physical stability, pH determination, content homogeneity examination, and in vitro medicine release analysis. Four formulations of Vemurafenib microemulsions successfully met all criteria in the microemulsion characterisation and assessment tests. The droplet sizes in these microemulsions fell inside the range of microemulsions, which is less than 200 nm. They were then used to create microemulsion-based hydrogels, employing Carbamer 340 as a gelling agent by conducting a simple mixing method. Hydrogels formed from microemulsions containing 1-Octyl-3-methylimidazolium hexafluorophosphate demonstrated the ability to form clear hydrogels with desirable consistency. Regarding Ex-vivo permeability study and skin deposition the permeability profiles of GOT3 formula exhibits a permeability measurement of 33569± 344 mP.s at 6 rpm, whereas GOT4 demonstrates a permeability measurement of 54723± 380 mP.s at 6 rpm. During the skin irritation test, there was no apparent erythema and edema were observed when compared to the negative group. This may indicate that the designed microemulsion-based gel formulation demonstrates good biocompatibility with skin tissue. Topical delivery of vemurafenib is a promising route of drug administration to melanoma skin. Ionic liquids (ILs) have permeation-enhancing properties with either hydrophilic or lipophilic characteristics‎‎‎‎‎.

Region-Specific Decellularization of Porcine Uterine Tube Extracellular Matrix: A New Approach for Reproductive Tissue-Engineering Applications

The uterine tube extracellular matrix is a key component that regulates tubal tissue physiology, and it has a region-specific structural distribution, which is directly associated to its functions. Considering this, the application of biological matrices in culture systems is an interesting strategy to develop biomimetic tubal microenvironments and enhance their complexity. However, there are no established protocols to produce tubal biological matrices that consider the organ morphophysiology for such applications. Therefore, this study aimed to establish region-specific protocols to obtain decellularized scaffolds derived from porcine infundibulum, ampulla, and isthmus to provide suitable sources of biomaterials for tissue-engineering approaches. Porcine uterine tubes were decellularized in solutions of 0.1% SDS and 0.5% Triton X-100. The decellularization efficiency was evaluated by DAPI staining and DNA quantification. We analyzed the ECM composition and structure by optical and scanning electronic microscopy, FTIR, and Raman spectroscopy. DNA and DAPI assays validated the decellularization, presenting a significative reduction in cellular content. Structural and spectroscopy analyses revealed that the produced scaffolds remained well structured and with the ECM composition preserved. YS and HEK293 cells were used to attest cytocompatibility, allowing high cell viability rates and successful interaction with the scaffolds. These results suggest that such matrices are applicable for future biotechnological approaches in the reproductive field.

Cloud Point Extraction Coupled to Flame Atomic Absorbance Spectroscopy for Cobalt (II) determination with Azo-Azomethine Dye

The azo-azomethine dye 6,6'-((1E,1'E)-((4-methyl-1,2-phenylene)bis(azanylylidene))bismethanylylidene))bis(2-methoxy-3-(o-tolyldiazenyl) phenol) (L3) were synthesized and characterized by UV-visible, FT-IR, 1H-NMR and Mass spectroscopy. The effect of the several factors on the CPE pre-concentration of Cobalt (II) with L3 was optimized. Extensive thermodynamic study has been presented to understand the mechanism of extraction and solubilization of studied complex in Triton X-100 micelles. Under the optimized conditions, enrichment factor of 54 is achieved leading to limit of detection and limit of quantitation of 11.5 and 38.4 ngmL-1 respectively. Under the optimal conditions the calibration plot is linear in the range of 0.025-3 μg mL-1, the precision (RSD%; n=8) of the proposed method is of 0.823% at 0.05 μg mL-1 of Co (II). This method is applied to the determination of Co (II) in various environmental samples.

Impacts of Short-Chain Alcohols on the Cloudy Development and Thermodynamics of Triton X-100 and Metformin Hydrochloride Drug Mixture

Impacts of Short-Chain Alcohols on the Cloudy Development and Thermodynamics of Triton X-100 and Metformin Hydrochloride Drug Mixture

Ionic liquids-based nanoemulsion for assisted by ultrasound liquid–liquid microextraction of UV-filters in water samples

Organic UV filters can contaminate various bodies of water such as streams, rivers, and seas, and as such, they have been identified as emerging contaminants of relevance in aquatic ecosystems. Therefore, the detection and determination of these compounds at low concentrations from an environmentally friendly perspective are necessary. In this work, an ionic liquids based nanoemulsion for the ultrasound assisted liquid–liquid microextraction (ILs-NE/US-LLME) of benzophenone-3, octocrylene, octisalate and octinoxate in different water bodies was reached. The material was based on a mixture of the ionic liquids 1,3-didecyl-2-methylimidazolium chloride and 1-butyl-3-methylimidazolium hexafluorophosphate, and the surfactant Triton X-100. The microextraction process was assisted by ultrasound and was performed using 475 µL of extractant solvent and 10.0 mL of sample, and an optimal preconcentration factor of 100 was achieved. The detection was performed by HPLC-UV in less than 5 min for all analytes. Low detection limits (lower than 2.0 µg L−1) and an optimal precision (RSD lower than 6.7 %) were obtained for the four UV filters. Different water samples (pool, stream, river, sea and a simulated sample) were analyzed and satisfactory recovery values were obtained in all cases (82–119 %). The method was fast (15 min−1) and it presented an optimal value of greenness (0.50) according to the AGREE metric. The extractant material was easily synthesized using a minimum amount of materials. The chromatographic procedure for separation and detection of the analytes was simple and fast. Moreover, the method was more environmentally friendly compared to previous work reported in the literature.

High-efficiency triplet-triplet annihilation upconversion microemulsion with facile preparation and decent air tolerance.

Current research of triplet-triplet annihilation upconversion (TTA-UC) faces difficulty such as overuse of organic solvents and quenching of excited triplet sensitizers by molecular oxygen. Herein, we propose an efficient and facile preparation strategy of TTA-UC microemulsion to overcome these issues. With simple device and short preparation process, air-stable TTA-UC with a high upconversion efficiency of 16.52% was achieved in microemulsion coassembled from TritonX114, tetrahydrofuran and upconverting chromophores (platinum octaethyl-porphyrin and 9,10-diphenylanthracene). This is comparable to the highest UC efficiency ever reported for TTA-UC microemulsion systems. The excellent UC performance of TX114-THF could be attributed to two perspectives. Firstly, small-size micelle accommodated chromophores up to high concentrations in organic phase, which promoted efficient molecular collision. Additionally, high absorbance at 532nm ensured full use of excitation light, getting more long wavelength photons involved in the TTA-UC process. Moreover, air-stable TTA-UC also performed well in microemulsion with various surfactants, including nonionic surfactants (Tween 20, Tween 80, Triton X-110, Triton X-114), ionic surfactants (sodium dodecyl sulfate, cetyltrimethyl ammonium bromide) and block copolymers (pluronic F127, pluronic P123), through three conjectural assembly models according to the structural characteristics of surfactant molecules (concentrated, uncompacted and scattered). These discoveries could provide estimable reference for selection of surfactants in relevant fields of TTA-UC.

Mechanisms of Triton X-100 reducing the Ag+-resistance of Enterococcus faecalis.

To investigate the mechanism of Triton X-100 (TX-100) reducing the Ag+-resistance of Enterococcus faecalis (E. faecalis), and evaluate the antibacterial effect of TX-100+Ag+ against the induced Ag+-resistant E. faecalis (AREf). The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of AgNO3 against E. faecalis with/without TX-100 were determined to verify the enhanced antibacterial activity. Transmission electron microscopy (TEM) was used to observe the morphological changes of E. faecalis after treatment. The intra- and extracellular concentration of Ag+ in treated E. faecalis was evaluated using inductively coupled plasma mass spectrometer (ICP-MS). The changes in cell membrane potential and integrity of treated E. faecalis were also observed using the flow cytometer. Moreover, AREf was induced through continuous exposure to sub-MIC of Ag+ and the antibacterial effect of TX-100+Ag+ on AREf was further evaluated. The addition of 0.04% TX-100 showed maximal enhanced antibacterial effect of Ag+ against E. faecalis. The TEM and ICP-MS results demonstrated that TX-100 could facilitate Ag+ to enter E. faecalis through changing the membrane structure and integrity. Flow cytometry further showed the effect of TX-100 on membrane potential and permeability of E. faecalis. In addition, the enhanced antibacterial effect of TX-100+Ag+ was also confirmed on induced AREf. TX-100 can facilitate Ag+ to enter E. faecalis through disrupting the membrane structure and changing the membrane potential and permeability, thus reducing the Ag+-resistance of E. faecalis and enhancing the antibacterial effect against either normal E. faecalis or induced AREf.

Practical considerations for birefringence microscopy of myelin structure: Microscope design and tissue processing for effective imaging

Abstract Despite the interest in studying and quantifying the structural integrity of myelin in postmortem brain tissue, current methods for high-resolution imaging of myelin with optical microscopy are not sufficient. While imaging methods must have adequate resolution and sensitivity to detect microstructural alterations to myelin that are relevant in aging and neurodegenerative disease, an equally critical aspect is to minimize myelin damage that is induced during tissue processing steps. Birefringence microscopy (BRM) is a powerful technique that leverages the structural anisotropy of myelin to provide detailed, label-free images of myelin at any diffraction-limited optical resolution, while maintaining a simple and low-cost setup. Building on our previous work, we have developed a new BRM system and image processing pipeline that enable efficient, high-throughput imaging of myelin structure at multiple scales. Here, we utilize this system to systematically assess the damage to myelin that is induced by several common tissue processing steps in brain sections from the rhesus monkey. Images taken of the same myelinated axons, before and after each tissue processing step, provide direct evidence that mishandling of tissue during sample preparation can cause significant structural alterations to myelin. First, we report on key advancements to our BRM system, imaging procedure, and image processing pipeline, which provide significant increases to the speed and efficiency of BRM. These include integrating fast piezoelectric rotational stages, minimizing the number of images required (to three images) for determining birefringence parameter maps, and implementing an analytical solution for directly determining birefringence parameter maps. Second, using this BRM system, we demonstrate that effective myelin imaging requires (1) the avoidance of prolonged drying or dehydration of tissue, (2) the selection of the optimal mounting medium (85% glycerol), (3) the avoidance of tissue permeabilization with detergents (i.e., Triton X-100 and Saponin), and (4) the selection of a suitable tissue-section thickness (15, 30 and 60 µm) based on the region of interest. In addition to serving as a guide for new users interested in imaging myelin, these basic experiments in sample preparation highlight that BRM is very sensitive to changes in the underlying lipid structure of myelin and suggest that optimized BRM can enable new studies of myelin breakdown in disease. In this work, we show that BRM is a leading method for detailed imaging and characterization of myelin, and we provide direct evidence that the structure of myelin is highly sensitive to damage during inadequate preparation of brain tissue for imaging, which has previously not been properly characterized for birefringence imaging of myelin. For the most effective, high-resolution imaging of myelin structure, tissue processing should be kept to a minimum, with sections prevented from dehydration and mounted in 85% glycerol. With proper preservation of myelin structure, BRM provides exquisitely detailed images that facilitate the assessment of myelin pathology associated with injury or disease.