Μm Depth Research Articles

Optimizing tissue clearing and imaging methods for human brain tissue.

ObjectivesTo identify optimum sample conditions for human brains, we compared the clearing efficiency, antibody staining efficiency, and artifacts between fresh and cadaver samples.MethodsFresh and cadaver samples were cleared using X-CLARITY™. Clearing efficiency and artifact levels were calculated using ImageJ, and antibody staining efficiency was evaluated after confocal microscopy imaging. Three staining methods were compared: 4-day staining (4DS), 11-day staining (11DS), and 4-day staining with a commercial kit (4DS-C). The optimum staining method was then selected by evaluating staining time, depth, method complexity, contamination, and cost.ResultsFresh samples outperformed cadaver samples in terms of the time and quality of clearing, artifacts, and 4′,6-diamidino-2-phenylindole (DAPI) staining efficiency, but had a glial fibrillary acidic protein (GFAP) staining efficiency that was similar to that of cadaver samples. The penetration depth and DAPI staining improved in fresh samples as the incubation period lengthened. 4DS-C was the best method, with the deepest penetration. Human brain images containing blood vessels, cell nuclei, and astrocytes were visualized three-dimensionally. The chemical dye staining depth reached 800 µm and immunostaining depth exceeded 200 µm in 4 days.ConclusionsWe present optimized sample preparation and staining protocols for the visualization of three-dimensional macrostructure in the human brain.

Monte Carlo calculation of Spencer-Attix and Bragg-Gray stopping-power ratios of tissue-to-air for ISO reference beta sources — an EGSnrc study

On-axis Spencer-Attix and Bragg-Gray stopping power ratios of tissue-to-air are calculated for 25 radiation fields of ISO beta sources 147Pm, 85Kr, 90Sr/90Y and 106Ru/106Rh using the EGSnrc-based Monte Carlo code system. These values are calculated at 5 and 70 μm depths in a unit density ICRU 4-element tissue phantom, separately, for beta and photon fields using the published phase-space data at different distances with or without beam flattening filter. The study shows that the calculated values of stopping power ratios of tissue-to-air based on Spencer-Attix cavity theory are higher than the values using Bragg-Gray cavity theory for the above-mentioned sources. For example, for 90Sr/90Y source with 3 mm/4 mm PMMA filter, depending upon cavity length (l = 0.025–0.25 cm), a comparison of Spencer-Attix theory-based values with the ISO-mentioned Bragg-Gray theory-based value of 1.110 shows differences up to 1.2%. Although the study has been done for both beta and photon fields, the data corresponding to electron phase-space data are relevant as the gamma dose to the total dose is insignificant.

Effect of deep rolling on subsurface conditions of CoCr28Mo6 wrought alloy to improve the wear resistance of endoprostheses

Wear of orthopaedic endoprostheses is associated with adverse local and systemic reactions and can lead to early implant failure. Manufacturing determines the initial subsurface microstructure of an alloy that influences the implant's wear behaviour. Therefore, this study aims at generating enhanced wear resistances by a modification of the surface and subsurface microstructure of a CoCr28Mo6 wrought alloy by applying deep rolling.The state of the art was investigated by means of eleven retrieved CoCr28Mo6 hip implant components from different manufacturers with respect to their subsurface microstructure and micro hardness profiles. CoCr28Mo6 wrought alloy samples (DIN EN ISO 5832–12) were aged at 750 °C for 24 h and/or plastically deformed by deep rolling with varying axial forces (170 N, 230 N and 250 N). The samples were metallographically prepared and investigated using optical and scanning electron microscopy with EDS and EBSD, micro hardness testing, XRD and tribological testing.The retrieved implant components revealed that, independent of the manufacturer, neither the head nor the stem trunnion exhibited a defined subsurface condition. The dominant phase within the implants was face-centered cubic (fcc). Some implants exhibited single hexagonal close-packed (hcp) grains due to a stress-induced phase transformation. The initial CoCr28Mo6 wrought alloy had a fcc crystal structure. After isothermal aging, the matrix entirely transformed to a hcp structure. In the initial fcc-condition, deep rolling generated a plastically deformed surface layer within the first 100 μm and stress-induced phase transformation to hcp was observed. Micro hardness gradients were present in the subsurface of up to 600 μm depth and exhibited a maximum increase of 34% by deep rolling in comparison to the initial fcc-matrix. This trend was confirmed by a correlated increase in residual compressive stresses. In tribological tests under serum lubrication, the modified samples generated lower wear in comparison to the contemporarily used fcc-matrix samples. This study demonstrates that deep rolling is an effective processing to modify the subsurface of a biomedical CoCr28Mo6 wrought alloy in order to increase the wear resistance. The intentional transformation from the fcc to the hcp phase induced by deformation offers great potential for implant application.

A Hybrid Microfluidic Differential Carbonator Approach for Enhancing Microalgae Growth: Inline Monitoring Through Optical Imaging

Microalga biomass is considered as a potential feedstock for renewable energy owing to its high lipid content. Conventional microalgae cell culturing methods mostly require lengthy procedures and involve laborious and time-consuming steps. On the other hand, microalgae culturing in microfluidics offers several advantages over conventional methods, such as high sensitivity, enhanced mass and heat transfer, reduced runtime and cost, lower consumption of reagent, and better interaction of species. In this study, initially, a novel hybrid microfluidic differential carbonator (µ-DC) platform was constructed in-house. Then, the performance of this system was tested by microalgae culturing. In this regard, Picochlorum sp. was selected as a model microalgae cell due to its fast growth and high lipid content. The operating conditions for the microalgae growth were 2–6% (vol) inlet CO2 concentration, 49 µmole m2/s light intensity, and 1:0.025 inlet ratio of algae to media. Both spatial and temporal changes of the microalgae growth were investigated. A maximum microalgae growth after 45 h was found to be $$3.37\times {10}^{10}$$ cells/mL in a 50 µm depth cell culture (µ-CC), which is higher cell density than literature values. Such inflate of cell density can be attributed to the fact in improving the mass transfer and providing better contact of microalgae with the ingredients of the photosynthesis process. This remarkable improvement in the growth of cells is of significant importance for many applications, especially for biodiesel production.

Numerical simulation and experimental study of pulsed CO2 laser-induced micro-channels on poly-ether sulfone (PES) polymer

TEA CO2 laser pulses were used to create micro-channels on PES samples with various low fluences and scanning speeds in 16–50 J/cm2 and 23–45 µm/s ranges, respectively. Different micro-channels were created with 142–342 µm widths and 85–440 µm depths which has been 3-dimensionally simulated using ABAQUS software based on the finite element method and CIN heat source. The sharp spike (~85 ns FWHM) and long tail (~3.8 µs) parts of the laser pulses were mathematically modeled with individual Gaussian profiles. A good agreement has been found between the simulated and experimental results. Namely, the maximum relative errors of the depth and opening width of the grooves remain below 11.5% and 12.3%, respectively. However, the corresponding mean values of these relative errors do not exceed 4.1% and 7.5%, respectively.

Depth- and direction-dependent changes in solute transport following cross-linking with riboflavin and UVA light in ex vivo porcine cornea

Diffusion is an important mechanism of transport for nutrients and drugs throughout the avascular corneal stroma. The purpose of this study was to investigate the depth- and direction-dependent changes in stromal transport properties and their relationship to changes in collagen structure following ultraviolet A (UVA)-riboflavin induced corneal collagen cross-linking (CXL). After cross-linking in ex vivo porcine eyes, fluorescence recovery after photobleaching (FRAP) was performed to measure fluorescein diffusion in the nasal-temporal (NT) and anterior-posterior (AP) directions at corneal depths of 100, 200, and 300 μm. Second harmonic generation (SHG) imaging was also performed at these three corneal depths to quantify fiber alignment. For additional confirmation, an electrical conductivity method was employed to quantify ion permeability in the AP direction in corneal buttons and immunohistochemistry (IHC) was used to image collagen structure. Cross-linked corneas were compared to a control treatment that received the riboflavin solution without UVA light (SHAM). The results of FRAP revealed that fluorescein diffusivity decreased from 23.39 ± 11.60 μm2/s in the SHAM group to 19.87 ± 10.10 μm2/s in the CXL group. This change was dependent on depth and direction: the decrease was more pronounced in the 100 μm depth (P = 0.0005) and AP direction (P = 0.001) when compared to the effect in deeper locations and in the NT direction, respectively. Conductivity experiments confirmed a decrease in solute transport in the AP direction (P < 0.0001). FRAP also detected diffusional anisotropy in the porcine cornea: the fluorescein diffusivity in the NT direction was higher than the diffusivity in the AP direction. This anisotropy was increased following CXL treatment. Both SHG and IHC revealed a qualitative decrease in collagen crimping following CXL. Analysis of SHG images revealed an increase in coherency in the anterior 200 μm of CXL treated corneas when compared to SHAM treated corneas (P < 0.01). In conclusion, CXL results in a decrease in stromal solute transport, and this decrease is concentrated in the most anterior region and AP direction. Solute transport in the porcine cornea is anisotropic, and an increase in anisotropy with CXL may be explained by a decrease in collagen crimping.

Ex vivo three-dimensional elemental imaging of mouse brain tissue block by laser-induced breakdown spectroscopy.

Measurement and reconstruction of an elemental image of large brain tissue will be beneficial to the diagnosis of neurological brain diseases. Herein, laser-induced breakdown spectroscopy (LIBS) is introduced for three dimensional (3D) elemental analysis of paraffin-embedded mouse brain tissue blocks. It is used for the first time towards the mapping of mouse brain block samples. A micro-LIBS prototype is developed for brain elemental imaging and a layer-by-layer approach is used to reconstruct the 3D distribution of Ca, Mg, Na, Cu, and P in the brain tissue. Images are captured with 50 μm lateral resolution and 300 μm depth resolution. The images show that the reclamation area of the cortex surface is enriched with Ca and Mg. In contrast, the Cu distribution is circular and is found primarily in the entirety of the cerebral cortex for the paraffin-embedded brain samples. Elemental imaging results suggest that the highest P intensity is found in the cerebellum nearby the middle sagittal plane in the left-brain paraffin block. These preliminary results indicate that LIBS is a potentially powerful tool for elemental bioimaging of the whole brain and may further improve the understanding of complex brain mechanisms.

High-Throughput Synthesis of Liposome Using an Injection-Molded Plastic Micro-Fluidic Device.

For mass production of liposomes, we designed a plastic micro-channel device on the basis of 5 μm of micro-nozzle array forming T-junction with 100 μm depth of micro-channel. A micro-channel unit for synthesizing liposomes consisted of two micro-nozzle arrays for mixing two solutions as well as delivery and recovery channels for supplying solutions and collecting liposome suspension. The number of micro-nozzles was approximately 2400 for a micro-channel unit, and seven units were applied independently on a micro-channel plate. The plastic micro-channel plate was injection-molded for mass production using a micro-channel stamper previously fabricated by UV lithography and nickel electroforming process. A plastic cover plate with seven pairs of inlet and outlet ports was machined by mechanical milling and drilling and was assembled with a micro-channel plate using a holder to form a liposome synthesizing device. Flow and mixing of solutions in the micro-channels were tested using colored water to check the micro-fluidic characteristics of the device. Finally, a L-α-phosphatidylcholine (SOY PC) liposome was synthesized using EtOH solution of SOY PC (95%) and saline (0.85% NaOH solution) to find that the liposomes were around 230 and 260 nm in diameter, depending on the flow rate of the lipid solution.

EXPERIMENTAL RESEARCH AND CFD SIMULATION OF CROSS FLOW MICROCHANNEL HEAT EXCHANGER

In this study, a cross flow microchannel heat exchanger has been manufactured out of standard sizes using aluminum material. The plate dimensions of heat exchangers have been 50x50x3 (mm3) that composed of two plates in cross flow arrangement. All evaluated geometries have been consisted of square microchannels with 490 μm width and 490 μm depth. An appropriate experimental facility has been established to perform the fluid flow and heat experiments. Moreover, heat transfer and fluid flow characteristics in microchannels have been simulated by ANSYS Fluent V15 Computer Program and experimental results have been compared with Computational Fluid Dynamics (CFD) results. Results showed that experimental heat transfer data was a very good agreement between data obtained by CFD simulation. However, the numeric pressure drop values have not been compatible with experimental ones.

Effect of Propolis Nanoparticles against Enterococcus faecalis Biofilm in the Root Canal.

To determine the antibacterial effect of propolis nanoparticles (PNs) as an endodontic irrigant against Enterococcus faecalis biofilm inside the endodontic root canal system. Two-hundred-ten extracted human teeth were sectioned to obtain 6 mm of the middle third of the root. The root canal was enlarged to an internal diameter of 0.9 mm. The specimens were inoculated with E. faecalis for 21 days. Following this, specimens were randomly divided into seven groups, with 30 dentinal blocks in each group including: group I—saline; group II—propolis 100 µg/mL; group III—propolis 300 µg/mL; group IV—propolis nanoparticle 100 µg/mL; group V—propolis nanoparticle 300µg/mL; group VI—6% sodium hypochlorite; group VII—2% chlorhexidine. Dentin shavings were collected at 200 and 400 μm depths, and total numbers of CFUs were determined at the end of one, five, and ten minutes. The non-parametric Kruskal–Wallis and Mann–Whitney tests were used to compare the differences in reduction in CFUs between all groups, and probability values of p < 0.05 were set as the reference for statistically significant results. The antibacterial effect of PNs as an endodontic irrigant was also assessed against E. faecalis isolates from patients with failed root canal treatment. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were also performed after exposure to PNs. A Raman spectroscope, equipped with a Leica microscope and lenses with curve-fitting Raman software, was used for analysis. The molecular interactions between bioactive compounds of propolis (Pinocembrin, Kaempferol, and Quercetin) and the proteins Sortase A and β-galactosidase were also understood by computational molecular docking studies. PN300 was significantly more effective in reducing CFUs compared to all other groups (p < 0.05) except 6% NaOCl and 2% CHX (p > 0.05) at all time intervals and both depths. At five minutes, 6% NaOCl and 2% CHX were the most effective in reducing CFUs (p < 0.05). However, no significant difference was found between PN300, 6% NaOCl, and 2% CHX at 10 min (p > 0.05). SEM images also showed the maximum reduction in E. faecalis with PN300, 6% NaOCl, and 2% CHX at five and ten minutes. CLSM images showed the number of dead cells in dentin were highest with PN300 compared to PN100 and saline. There was a reduction in the 484 cm−1 band and an increase in the 870 cm−1 band in the PN300 group. The detailed observations of the docking poses of bioactive compounds and their interactions with key residues of the binding site in all the three docking protocols revealed that the interactions were consistent with reasonable docking and IFD docking scores. PN300 was equally as effective as 6% NaOCl and 2% CHX in reducing the E. faecalis biofilms.

Design, manufacture and application of a microreactor for the decomposition of ethyl mercaptan on an H-ZSM-5 catalyst

Catalytic decomposition of the ethyl mercaptan over an H-ZSM-5 zeolite in a newly designed microreactor was undertaken in this study. A numerically developed distributor possessing superior flow distribution was amended to 24 parallel microchannels with 64 mm length, 700 μm width, and 600 μm depth. Ethyl mercaptan complete conversion required 350 °C through the developed microreactor while in a conventional fixed-bed-reactor 400 °C was needed. Higher selectivity towards the main products of this decomposition (hydrogen sulfide and ethylene) were observed in this microreactor in comparison to that of a fixed-bed-reactor. In addition, lower selectivity towards formation of byproducts was displayed in the former. Investigation of the WHSV variations upon the product distribution through this microreactor revealed that, conversion of the ethyl mercaptan was reduced to 60 from the 89% through enhancement of the feed rate from 0.32 to 0.48 h−1.

Pulsatile Flow-Induced Fatigue-Resistant Photopolymerizable Hydrogels for the Treatment of Intracranial Aneurysms.

An alternative intracranial aneurysm embolic agent is emerging in the form of hydrogels due to their ability to be injected in liquid phase and solidify in situ. Hydrogels have the ability to fill an aneurysm sac more completely compared to solid implants such as those used in coil embolization. Recently, the feasibility to implement photopolymerizable poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogels in vitro has been demonstrated for aneurysm application. Nonetheless, the physical and mechanical properties of such hydrogels require further characterization to evaluate their long-term integrity and stability to avoid implant compaction and aneurysm recurrence over time. To that end, molecular weight and polymer content of the hydrogels were tuned to match the elastic modulus and compliance of aneurysmal tissue while minimizing the swelling volume and pressure. The hydrogel precursor was injected and photopolymerized in an in vitro aneurysm model, designed by casting polydimethylsiloxane (PDMS) around 3D printed water-soluble sacrificial molds. The hydrogels were then exposed to a fatigue test under physiological pulsatile flow, inducing a combination of circumferential and shear stresses. The hydrogels withstood 5.5 million cycles and no significant weight loss of the implant was observed nor did the polymerized hydrogel protrude or migrate into the parent artery. Slight surface erosion defects of 2–10 μm in depth were observed after loading compared to 2 μm maximum for non-loaded hydrogels. These results show that our fine-tuned photopolymerized hydrogel is expected to withstand the physiological conditions of an in vivo implant study.

A biobased self-healing thermoset coating with a dynamic photosensitive molecule

A biobased photo-healable thermoset coating has been developed from 4 components in only 2 steps via a solvent-free process involving an epoxy/amine reaction. Its composition includes epoxidized linseed oil and a dynamic molecule (4,4′-diaminodiphenyl disulfide) which, under UV irradiation, can be photolyzed reversibly. Compared to an analogous material without disulfide bond, this coating exhibits a lower crosslinking density and a higher elongation at break. Importantly, a 100 μm depth scratch has been significantly repaired under UV light: a photo-healable coating having both thermoplastic and thermosetting properties has thus been elaborated.

Radiosensitization by Au-nanofilm at micrometer level using confocal Raman spectroscopy.

To measure the radiosensitization by an Au-nanofilm (GNF) at a micrometer level on a radiochromic film (RCF) using confocal Raman spectroscopy (CRS). Unlaminated radiochromic films were irradiated by 200kVp x-ray from 0.3 to 50Gy to obtain a calibration curve. Raman spectra of these films were measured by positioning the postirradiated RCF perpendicular to the CRS monochromatic beam and reading a depth profile of the film along the lateral axis. The Raman peak corresponding to the C≡C peak was obtained from a region of interest of 100×5µm2 . To investigate the radiosensitization by GNF, two sets of RCF, one attached to a 100-nm thick GNF and the other without GNF were irradiated at 0.5Gy by 50 and 120kVp X-rays. The spatial resolution of the CRS on the RCF was quantified by the modulation transfer function method (MTF). Thus, in the spatial resolution determined by MTF, the doses deposited on the films were evaluated. The dose enhancement factor (DEF) was obtained in the measurable micro-size by comparing doses deposited on the RCFs with and without GNF. To verify the experimental results, Monte Carlo simulations following the experimental set up were performed using Geant4. In addition, analytical calculations for the radiosensitization by GNF were carried out. The confocal Raman spectroscopy on the RCF achieved a spatial resolution of ~6μm. An experimental DEF within the first 6μm depth from the surface of RCF was found to be 17.9 for 50kVp and 14.7 for 120kVp. The DEF for the same depth obtained by MC and analytical calculations was 13.53 and 9.75 for 50kVp, and 10.63 and 6.67 for 120kVp, respectively. The experimental DEF as a function of the distance from GNF was consistent with data from previous studies and the MC simulations, supporting that CRS in conjunction with the RCF is a feasible micrometer-resolution dosimeter.

Preclinical study in a corneal bioreactor of the effectiveness of amniotic membrane of umbilical cord extract (AMUCE) for the treatment of corneal deep stromal ulcerations

AbstractPurposeOur patented corneal bioreactor (BR) improves the long‐term survival of human corneas ex vivo by restoring intraocular pressure and renewal of storage medium.AimTo use the BR to test the efficacy of eye drops containing devitalized ground amniotic membrane of umbilical cord extract (AMUCE) to treat deep stromal ulcerations. AMUCE contains high level of proteoglycans, collagen and physiological growth factors.MethodsOnly pairs of human corneas (with ECD &gt; 800 cells/mm2) were used to obtain best possible controls. They were first stored for 2 weeks into the BR filled with CorneaMax (Eurobio, France), with 21 mmHg in the endothelial chamber to allow regeneration of a normal epithelium. Then, an 8.0 mm diameter calibrated epithelial ulcer was made with a spatula and a 6 mm diameter × 100 µm in depth was made with Excimer laser in the stroma. Using a specific BR lid allowing sterile handling, each cornea was treated topically 4 times daily either by the AMUCE eye drops (TBF, France) or by the vehicle (0.9% NaCl). Closure of the ulceration was monitored daily using fluorescein staining quantified by image analysis (FIJI) of macro‐zoom microscopy. After wound healing corneas were fixed for histology (HES) and immunolabeling of epithelial markers.ResultsThree pairs were used. Epithelium wound healing was obtained at D3, D9, D10 with AMUCE and D3, D9, D19 with the vehicle. Histology performed in ulcerations centre showed 3‐6 epithelial layers with AMUCE and 1‐4 with the vehicle. At D21, significant epithelial detachment was observed only for 2 corneas treated with the vehicle. For one pair, immunolabelling for E‐cadherin and Cytokeratin K3/K12 showed better epithelial differentiation with AMUCE than with vehicle. No evidence of stromal regeneration was observed in the 6 corneas.ConclusionsAMUCE has potential for a faster and lasting epithelium regeneration (stratification and maturation). Nevertheless, it has no impact on stromal regeneration in this model.

On the microstructure evolution of a nuclear-use steel after heavy ion irradiation

A nuclear-use ferritic-martensitic steel was irradiated by 196 MeV Kr ions. Both the low damage level area and the high damage level area were investigated by a transmission electron microscope (TEM), to reveal the irradiated microstructural features. In low damage level area ranging from surface to 6 μm depth, there were a few dislocation loops and black dots induced by Kr irradiation. The type of dislocation loops could be primarily ao<100> or ao/2<100> type. In addition, it was found that the sink effect of grain boundaries and stability of small precipitates were evident.

Металл-стимулированное разложение поверхности сапфира в потоке электронов с энергией 70 keV

Metal-stimulated decomposition of sapphire surface at room temperature in a flux of electrons with energy of 70 keV. The stimulant used is an ensemble of crystalline islands of gold. It was first established that the presence of an ensemble of gold islands on the surface of sapphire significantly reduces substrate temperature and electron energy at which decomposition occurs surfaces. Etching pits form near gold islands and have an elongated shape with the following dimensions: length up to 1.2 μm, width up to 0.8 μm and average depth 20 nm. Maximum area the base of the islands increases from 0.6 to 1.8 μm2.

Performance study of dimple and channel textured tools during turning of Inconel-718

Alterations in cutting tool surfaces are being looked at as futuristic developments in cutting tool technology. Tool surface texturing by subtractive as well as an additive way can contribute to the tool performance and productivity improvement. The tool textures can be in the range of nano to micro-level. They help to reduce friction, manage the heat-induced during machining and facilitate chip removal. For textured surfaces, small cavities or dimples uniformly distributed over the sliding surface have been found to have the best tribological effect. In this study, two different micro-texture patterns i.e., micro-dimple and micro-channel were made on the rake-face close to the main cutting edge of the carbide tool insert. Ultra-short pulsed (nanosecond) fiber laser has been used for texturing. An array of texture patterns with 80 µm width/diameter and 70 µm depth has been engraved on the uncoated tungsten carbide tool. The textured inserts have been used for machining trials at 30 m/min to 100 m/min for Inconel 718 to compare the relative performance of the textures in terms of three components of cutting force and machined surface roughness. The experimental observations showed higher components of cutting force for channel textured tools in comparison to dimple textured tools. However, a better surface finish has been observed with channel textured tools. Cutting forces has been observed varying significantly with cutting speed in the case of dimple textured tools. Radial forces were observed higher in magnitude followed by tangential cutting force and feed force for both the textured tools. This study suggests having further studies considering the effect of different texture patterns on the cutting forces, surface roughness, and tool life while turning Inconel 718.

Investigating the influence of block copolymer micelle length on cellular uptake and penetration in a multicellular tumor spheroid model.

The efficient penetration of drug nanocarriers into tumors is an important prerequisite for therapeutic and diagnostic success. The physicochemical properties of nanocarriers, including size, shape and surface chemistry have been shown to influence their transport in biological systems. Recent studies have shown that elongated nanoparticles (NPs) can exhibit advantageous properties in comparison to spherical NPs, but these experiments have involved a variety of different materials, many of which are characterized by a broad size distribution. Here we describe a series of rigid rod-like micelles of uniform width, with narrow length distributions, and common surface chemistry, and examine their cell uptake and penetration into multicellular tumor spheroids (MCTSs) formed from two human breast cancer cell lines (MDA-MB-436 and MDB-MB-231). These micelles were prepared from a polyferrocenylsilane (PFS) diblock copolymer (BCP) with a corona block consisting of a statistical polymer of aminopropyl methacrylamide and oligo(ethyleneglycol methacrylate) (PFS27-b-PAPMA3-stat-POEGMA48). The rigid rod micelles, with a common width (12 nm) and lengths ranging from 80 to 2000 nm, were prepared by seeded growth crystallization-driven self-assembly in ethanol and then transferred to water. To consider whether changing the shape of these micelles affects its uptake and penetration behavior, analogous spherical micelles were prepared by direct nanoprecipitation into water. Both micelle shape and length were found to influence cellular uptake and penetration into 500 μm MCTSs. Laser confocal fluorescence microscopy was used to examine penetration of these micelles into three-dimensional MCTS up to 90 μm depth. Micelles with an elongated shape and short length (80 nm) demonstrated the deepest penetration into the MCTSs formed by MDA-MB-436 cells. Micelles with lengths of 200 nm also showed substantial penetration into these MCTS, but the extent and depth of tumor penetration of the rod-like micelles decreased with increasing aspect ratio. MCTS of MDA-MB-231 cells had a less dense, more open structure than those formed by MDA-MB-436 cells. Here more extensive penetration was observed, particularly for the longer micelle samples.

Protocol for Sampling Sequential Fin Spine Growth Intervals for Isotope Analysis in the Atlantic Bluefin Tuna

BackgroundMicromilled fish otoliths (ear bones) have been widely used for stable oxygen (δ18O) and carbon (δ13C) isotope analysis. The first dorsal fin spine is routinely used for ageing in the eastern Atlantic bluefin tuna (ABFT) population; however, stable isotope analysis remains unapplied in this hard structure. The objective of the present protocol is to achieve a sequential sampling of growth layers in the ABFT fin spine at high spatial resolution and along growth trajectory for stable isotope analysis.MethodsWe used a micromilling and micro-powder collecting technique for sequentially sampling annual growth layers of the fin spine bone collected from freshly caught ABFT. We assessed the carbonate content and the optimal drilling amount to ensure enough powder quantity was recovered from each annual growth band to accommodate accurate measurement of the δ18O and δ13C values.ResultsThe optimal drilling path included 20 drilling lines in 49 μm, with 400 μm depth and 900 μm-line width, which represents a time resolution of about 2.5 months. The minimum powder quantity required from each annual growth layer for δ18O and δ13C isotope analysis was approximately 180 μg because the carbonate contents in the fin spine powder was 5–6%. The δ18O profile assayed in the last growth intervals coincide with the surface δ18Oseawater for the southern west waters off the Norwegian coast, where the tuna was caught. The fluctuating pattern in the second growth interval may reflect certain fidelity to the Norwegian SE and Swedish SW coast and/or otherwise to the US west coast. The δ13Cspine displayed a larger temporal variability along the growth transect that may be explained by several factors including differences in habitat use, behavior, and even trophic level.ConclusionBased on the result obtained, we present an optimal standard protocol for the sampling of sequential, annually formed growth layers in the fin spine bone for stable isotope analysis using the micro-milling as a high precision technique. This protocol is particularly useful in endangered and/or protected species for which fin spines represent a non-lethal alternative to otoliths opening new research avenues to improve their management and conservation.