Optimization of sample preparation, fluorescence- and Raman techniques for environmental microplastics.

Microspectroscopic imaging techniques based on spontaneous Raman scattering, Stimulated Raman Scattering (SRS), or fluorescence (with a selective dye) can be used to detect environmental microplastics (MPs) and determine their chemical as well as physical properties. The present study first focuses on optimizing the sample preparation, including a new design for a density separation apparatus and optimization of the Nile Red staining procedure. Tests were carried out with both white and colored reference materials, as well as environmental MPs in a suspended matter sample from the Rhine river. The new 'MESSY' system has a mean recovery of 95 ± 5.5 % (three polymer materials, in duplicate). The optimized Nile Red staining allows coarse categorization of MPs into "polar" vs. "non-polar" materials based on their Fluorescence Index (emission wavelength), but fluorescent additives in the polymer can cause misclassification. For unambiguous identification of the polymer type, Raman spectroscopy can be used. Even colored polymers, with or without Nile Red staining, were readily identified by Raman spectroscopy using a red laser (785 nm), except for particles containing carbon black. A Deep-UV Raman microscope (ex = 248.6 nm) was constructed, which allowed identification of all colored plastics, even those pigmented with carbon black. Since unsupervised mapping with spontaneous Raman is very slow, point measurements are preferably used after preselection of particles of interest based on fluorescence imaging. SRS is several orders of magnitude faster than spontaneous Raman mapping but requires multiple scans at different z-heights and at multiple wavenumber settings to detect and identify all particles. The results are expected to contribute to the development of suitable methodologies for the detection and identification of environmental microplastics.

A versatile Raman setup with time-gating and fast wide-field imaging capabilities.

Raman spectroscopy is a well-established method for chemical identification, with a wide variety of applications. The two major limitations are that fluorescence can hamper detection, and that Raman imaging is slow; it typically takes multiple hours to measure even a small surface area. We have developed a multimodal setup that mitigates these limitations. The setup has a point-scanning mode that allows for time-gated as well as continuous Raman spectroscopy, and both modes use an 80 MHz, 532 nm excitation laser with up to 20 W of power. The fluorescence suppression capabilities of the setup were demonstrated by comparing time-gated to continuous detection of a Dracaena leaf. Raman bands showed a 4-8 times improvement in signal-to-background ratio, and one band that was invisible in the continuous measurement, became visible in the time-gated measurement. The setup also has a 4-band simultaneously detected wide-field mode. Using a set of beam splitters, the Raman signal from the sample is split. This signal is imaged onto four separate cameras, each with a specific band-pass filter. The wide-field data were processed using principal component analysis with k-means clustering. To illustrate the wide-field capabilities of the setup, a 1mm2 sample containing aspirin, caffeine and paracetamol was measured using 10 W excitation power. A 10-second measurement enabled identification of the compounds, and a 1-second measurement showed promising results. This brings the setup close to real-time imaging, showing great potential for applications in quality control or for measuring samples that change over time.

Multi-Parameter Analysis of Nanoplastics in Flow: Taking Advantage of High Sensitivity and Time Resolution Enabled by Stimulated Raman Scattering.

Here, we demonstrate the detection of nanoplastics (NPLs) in flow with stimulated Raman scattering (SRS) for the first time. NPLs (plastic particles <1000 nm) have recently been detected in different environmental samples and personal care products. However, their characterization is still an analytical challenge. Multiple parameters, including size, chemical composition, and concentration (particle number and mass), need to be determined. In an earlier paper, online field flow fractionation (FFF)-Raman analysis with optical trapping was shown to be a promising tool for the detection of particles in this size range. SRS, which is based on the enhancement of a vibrational transition by the matching energy difference of two laser beams, would allow for much more sensitive detection and, hence, much shorter acquisition times compared to spontaneous Raman microspectroscopy (RM). Here, we show the applicability of SRS for the flow-based analysis of individual, untrapped NPLs. It was possible to detect polyethylene (PE), polystyrene (PS), and poly(methyl methacrylate) (PMMA) beads with diameters of 100-5000 nm. The high time resolution of 60.5 µs allows us to detect individual signals per particle and to correlate the number of detected particles to the injected mass concentration. Furthermore, due to the high time resolution, optically trapped beads could be distinguished from untrapped beads by their peak shapes. The SRS wavenumber settings add chemical selectivity to the measurement. Whereas optical trapping is necessary for the flow-based detection of particles by spontaneous RM, the current study demonstrates that SRS can detect particles in a flow without trapping. Additionally, the mean particle size could be estimated using the mean width (duration) and intensity of the SRS signals.

Raman Diffusion-Ordered Spectroscopy.

The Stokes-Einstein relation, which relates the diffusion coefficient of a molecule to its hydrodynamic radius, is commonly used to determine molecular sizes in chemical analysis methods. Here, we combine the size sensitivity of such diffusion-based methods with the structure sensitivity of Raman spectroscopy by performing Raman diffusion-ordered spectroscopy (Raman-DOSY). The core of the Raman-DOSY setup is a flow cell with a Y-shaped channel containing two inlets: one for the sample solution and one for the pure solvent. The two liquids are injected at the same flow rate, giving rise to two parallel laminar flows in the channel. After the flow stops, the solute molecules diffuse from the solution-filled half of the channel into the solvent-filled half at a rate determined by their hydrodynamic radius. The arrival of the solute molecules in the solvent-filled half of the channel is recorded in a spectrally resolved manner by Raman microspectroscopy. From the time series of Raman spectra, a two-dimensional Raman-DOSY spectrum is obtained, which has the Raman frequency on one axis and the diffusion coefficient (or equivalently, hydrodynamic radius) on the other. In this way, Raman-DOSY spectrally resolves overlapping Raman peaks arising from molecules of different sizes. We demonstrate Raman-DOSY on samples containing up to three compounds and derive the diffusion coefficients of small molecules, proteins, and supramolecules (micelles), illustrating the versatility of Raman-DOSY. Raman-DOSY is label-free and does not require deuterated solvents and can thus be applied to samples and matrices that might be difficult to investigate with other diffusion-based spectroscopy methods.

Nanoscale Thermometry of Plasmonic Structures via Raman Shifts in Copper Phthalocyanine.

Temperature measurements at the nanoscale are vital for the application of plasmonic structures in medical photothermal therapy and materials science but very challenging to realize in practice. In this work, we exploit a combination of surface-enhanced Raman spectroscopy together with the characteristic temperature dependence of the Raman peak maxima observed in ß-phase copper phthalocyanine (ß-CuPc) to measure the surface temperature of plasmonic gold nanoparticles under laser irradiation. We begin by measuring the temperature-dependent Raman shifts of the three most prominent modes of ß-CuPc films coated on an array of Au nanodisks over a temperature range of 100-500 K. We then use these calibration curves to determine the temperature of an array of Au nanodisks irradiated with varying laser powers. The extracted temperatures agree quantitatively with the ones obtained via numerical modeling of electromagnetic and thermodynamic properties of the irradiated array. Thin films of ß-CuPc display low extinction coefficients in the blue-green region of the visible spectrum as well as exceptional thermal stability, allowing a wide temperature range of operation of our Raman thermometer, with minimal optical distortion of the underlying structures. Thanks to the strong thermal response of the Raman shifts in ß-CuPc, our work opens the opportunity to investigate photothermal effects at the nanoscale in real time.

Baker Grade IV Capsular Contracture Is Correlated with an Increased Amount of Silicone Material: An Intrapatient Study.

BACKGROUND: Breast implant surgery is one of the most frequently performed procedures by plastic surgeons worldwide. However, the relationship between silicone leakage and the most common complication, capsular contracture, is far from understood. This study aimed to compare Baker grade I with Baker grade IV capsules regarding their silicone content in an intradonor setting, using two previously validated imaging techniques. METHODS: Twenty-two donor-matched capsules from 11 patients experiencing unilateral complaints were included after bilateral explantation surgery. All capsules were examined using both stimulated Raman scattering (SRS) imaging and staining with modified oil red O (MORO). Evaluation was done visually for qualitative and semiquantitative assessment and automated for quantitative analysis. RESULTS: Using both SRS and MORO techniques, silicone was found in more Baker grade IV capsules (eight of 11 and 11 of 11, respectively) than in Baker grade I capsules (three of 11 and five of 11, respectively). Baker grade IV capsules also showed significantly more silicone content compared with the Baker grade I capsules. This was true for semiquantitative assessment for both SRS and MORO techniques ( P = 0.019 and P = 0.006, respectively), whereas quantitative analysis proved to be significant for MORO alone ( P = 0.026 versus P = 0.248 for SRS, respectively). CONCLUSIONS: In this study, a significant correlation between capsule silicone content and capsular contracture is shown. An extensive and continued foreign body response to silicone particles is likely to be responsible. Considering the widespread use of silicone breast implants, these results affect many women worldwide and warrant a more focused research effort. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Softness of hydrated salt crystals under deliquescence.

Deliquescence is a first-order phase transition, happening when a salt absorbs water vapor. This has a major impact on the stability of crystalline powders that are important for example in pharmacology, food science and for our environment and climate. Here we show that during deliquescence, the abundant salt sodium sulfate decahydrate, mirabilite (Na2SO4·10H2O), behaves differently than anhydrous salts. Using various microscopy techniques combined with Raman spectroscopy, we show that mirabilite crystals not only lose their facets but also become soft and deformable. As a result, microcrystals of mirabilite simultaneously behave crystalline-like in the core bulk and liquid-like at the surface. Defects at the surface can heal at a speed much faster than the deliquescence rate by the mechanism of visco-capillary flow over the surface. While magnesium sulfate hexahydrate (MgSO4⋅6H2O) behaves similarly during deliquescence, a soft and deformable state is completely absent for the anhydrous salts sodium chloride (NaCl) and sodium sulfate thenardite (Na2SO4). The results highlight the effect of crystalline water, and its mobility in the crystalline structure on the observed softness during deliquescence. Controlled hydrated salts have potential applications such as thermal energy storage, where the key parameter is relative humidity rather than temperature.

Liquid Core Waveguide Cell with In Situ Absorbance Spectroscopy and Coupled to Liquid Chromatography for Studying Light-Induced Degradation.

In many areas, studying photostability or the mechanism of photodegradation is of high importance. Conventional methods to do so can be rather time-consuming, laborious, and prone to experimental errors. In this paper we evaluate an integrated and fully automated system for the study of light-induced degradation, comprising a liquid handler, an irradiation source and exposure cell with dedicated optics and spectrograph, and a liquid chromatography (LC) system. A liquid core waveguide (LCW) was used as an exposure cell, allowing efficient illumination of the sample over a 12 cm path length. This cell was coupled to a spectrograph, allowing in situ absorbance monitoring of the exposed sample during irradiation. The LCW is gas-permeable, permitting diffusion of air into the cell during light exposure. This unit was coupled online to LC with diode array detection for immediate and automated analysis of the composition of the light-exposed samples. The analytical performance of the new system was established by assessing linearity, limit of detection, and repeatability of the in-cell detection, sample recovery and carryover, and overall repeatability of light-induced degradation monitoring, using riboflavin as the test compound. The applicability of the system was demonstrated by recording a photodegradation time profile of riboflavin.

Characterization of a liquid-core waveguide cell for studying the chemistry of light-induced degradation.

Many organic compounds undergo changes under the influence of light. This might be beneficial in, for example, water purification, but undesirable when cultural-heritage objects fade or when food ingredients (e.g., vitamins) degrade. It is often challenging to establish a strong link between photodegradation products and their parent molecules due to the complexity of the sample. To allow effective study of light-induced degradation (LID), a low-volume exposure cell was created in which solutes are efficiently illuminated (especially at low concentrations) while simultaneously analysed by absorbance spectroscopy. The new LID cell encompasses a gas-permeable liquid-core waveguide (LCW) connected to a spectrograph allowing collection of spectral data in real-time. The aim of the current study was to evaluate the overall performance of the LID cell by assessing its transmission characteristics, the absolute photon flux achieved in the LCW, and its capacity to study solute degradation in presence of oxygen. The potential of the LID set-up for light-exposure studies was successfully demonstrated by monitoring the degradation of the dyes eosin Y and crystal violet.

Multimodal, label-free fluorescence and Raman imaging of amyloid deposits in snap-frozen Alzheimer's disease human brain tissue.

Alzheimer's disease (AD) neuropathology is characterized by hyperphosphorylated tau containing neurofibrillary tangles and amyloid-beta (Aß) plaques. Normally these hallmarks are studied by (immuno-) histological techniques requiring chemical pretreatment and indirect labelling. Label-free imaging enables one to visualize normal tissue and pathology in its native form. Therefore, these techniques could contribute to a better understanding of the disease. Here, we present a comprehensive study of high-resolution fluorescence imaging (before and after staining) and spectroscopic modalities (Raman mapping under pre-resonance conditions and stimulated Raman scattering (SRS)) of amyloid deposits in snap-frozen AD human brain tissue. We performed fluorescence and spectroscopic imaging and subsequent thioflavin-S staining of the same tissue slices to provide direct confirmation of plaque location and correlation of spectroscopic biomarkers with plaque morphology; differences were observed between cored and fibrillar plaques. The SRS results showed a protein peak shift towards the ß-sheet structure in cored amyloid deposits. In the Raman maps recorded with 532 nm excitation we identified the presence of carotenoids as a unique marker to differentiate between a cored amyloid plaque area versus a non-plaque area without prior knowledge of their location. The observed presence of carotenoids suggests a distinct neuroinflammatory response to misfolded protein accumulations.

Label-free stimulated Raman scattering imaging reveals silicone breast implant material in tissue.

Millions of women worldwide have silicone breast implants. It has been reported that implant failure occurs in approximately a tenth of patients within 10 years, and the consequences of dissemination of silicone debris are poorly understood. Currently, silicone detection in histopathological slides is based on morphological features as no specific immunohistochemical technique is available. Here, we show the feasibility and sensitivity of stimulated Raman scattering (SRS) imaging to specifically detect silicone material in stained histopathological slides, without additional sample treatment. Histology slides of four periprosthetic capsules from different implant types were obtained after explantation, as well as an enlarged axillary lymph node from a patient with a ruptured implant. SRS images coregistered with bright-field images revealed the distribution and quantity of silicone material in the tissue. Fast and high-resolution imaging of histology slides with molecular specificity using SRS provides an opportunity to investigate the role of silicone debris in the pathophysiology of implant-linked diseases.

The search for a unique Raman signature of amyloid-beta plaques in human brain tissue from Alzheimer's disease patients.

Definite Alzheimer's disease (AD) diagnosis is commonly done on ex vivo brain tissue using immuno-histochemical staining to visualize amyloid-beta (Aß) aggregates, also known as Aß plaques. Raman spectroscopy has shown its potential for non-invasive and label-free determination of bio-molecular compositions, aiding the post-mortem diagnosis of pathological tissue. Here, we investigated whether conventional Raman spectroscopy could be used for the detection of amyloid beta deposits in fixed, ex vivo human brain tissue, taken from the frontal cortex region. We examined the spectra and spectral maps of three severe AD cases and two healthy control cases and compared their spectral outcome among each other as well as to recent results in the literature obtained with various spectroscopic techniques. After hyperspectral Raman mapping, Aß plaques were visualized using Thioflavin-S staining on the exact same tissue sections. As a result, we show that tiny diffuse or tangled-like morphological structures, visible under microscopic conditions on unstained tissue and often but erroneously assumed to be deposits of Aß, are instead usually an aggregation of highly auto-fluorescent lipofuscin granulates without any, or limited, plaque or plaque-like association. The occurrence of these auto-fluorescent particles is equally distributed in both AD and healthy control cases. Therefore, they cannot be used as possible criteria for Alzheimer's disease diagnosis. Furthermore, a unique plaque-specific/Aß spectrum could not be determined even after possible spectral interferences were carefully removed.

Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation.

High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases.

Circular Spectropolarimetric Sensing of Vegetation in the Field: Possibilities for the Remote Detection of Extraterrestrial Life.

Homochirality is a generic and unique property of all biochemical life, and the fractional circular polarization of light it induces therefore constitutes a potentially unambiguous biosignature. However, while high-quality circular polarimetric spectra can be easily and quickly obtained in the laboratory, accurate measurements in the field are much more challenging due to large changes in illumination and target movement. In this study, we measured various targets in the field, up to distances of a few kilometers, using the dedicated circular spectropolarimeter TreePol. We show how photosynthetic life can readily be distinguished from abiotic matter. We underline the potential of circular polarization signals as a remotely accessible means to characterize and monitor terrestrial vegetation, for example, for agriculture and forestry. In addition, we discuss the potential of circular polarization for the remote detection of extraterrestrial life.

Circular spectropolarimetric sensing of higher plant and algal chloroplast structural variations.

Photosynthetic eukaryotes show a remarkable variability in photosynthesis, including large differences in light-harvesting proteins and pigment composition. In vivo circular spectropolarimetry enables us to probe the molecular architecture of photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological and structural information. In the present study, we have measured the circular polarizance of several multicellular green, red, and brown algae and higher plants, which show large variations in circular spectropolarimetric signals with differences in both spectral shape and magnitude. Many of the algae display spectral characteristics not previously reported, indicating a larger variation in molecular organization than previously assumed. As the strengths of these signals vary by three orders of magnitude, these results also have important implications in terms of detectability for the use of circular polarization as a signature of life.

Imaging linear and circular polarization features in leaves with complete Mueller matrix polarimetry.

Spectropolarimetry of intact plant leaves allows to probe the molecular architecture of vegetation photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological information. In addition to the molecular signals due to the photosynthetic machinery, the cell structure and its arrangement within a leaf can create and modify polarization signals. Using Mueller matrix polarimetry with rotating retarder modulation, we have visualized spatial variations in polarization in transmission around the chlorophyll a absorbance band from 650 nm to 710 nm. We show linear and circular polarization measurements of maple leaves and cultivated maize leaves and discuss the corresponding Mueller matrices and the Mueller matrix decompositions, which show distinct features in diattenuation, polarizance, retardance and depolarization. Importantly, while normal leaf tissue shows a typical split signal with both a negative and a positive peak in the induced fractional circular polarization and circular dichroism, the signals close to the veins only display a negative band. The results are similar to the negative band as reported earlier for single macrodomains. We discuss the possible role of the chloroplast orientation around the veins as a cause of this phenomenon. Systematic artefacts are ruled out as three independent measurements by different instruments gave similar results. These results provide better insight into circular polarization measurements on whole leaves and options for vegetation remote sensing using circular polarization.

Screening for microplastics in sediment, water, marine invertebrates and fish: Method development and microplastic accumulation.

Measurements of microplastics in biota and abiotic matrices are key elements of exposure and risk assessments for this emerging environmental pollutant. We investigated the abundance of microplastics in field-collected biota, sediment and water. An improved sediment extraction method, based on density separation was developed. For analysis of microplastics in biota we found that an adapted enzymatic digestion protocol using proteinase K performed best, with a 97% recovery of spiked plastic particles and no observed degradation effects on the plastics in subsequent Raman analysis. Field analysis revealed that 8 of 9 tested invertebrate species from the North Sea and 68% of analyzed individuals of brown trout (Salmo trutta) from the Swedish West Coast had microplastics in them. Based on the number of plastic particles per kg d.w. the microplastic concentrations found in mussels were approximately a thousand-fold higher compared to those in sediment and surface water samples from the same location.

Fast High-Resolution Screening Method for Reactive Surfaces by Combining Atomic Force Microscopy and Surface-Enhanced Raman Scattering.

A fast high-resolution screening method for reactive surfaces is presented. Atomic force microscopy (AFM) and surface-enhanced Raman spectroscopy (SERS) are combined in one method in order to be able to obtain both morphological and chemical information about processes at a surface. In order to accurately align the AFM and SERS images, an alignment pattern on the substrate material is exploited. Subsequent SERS scans with sub-micron resolution are recorded in 30 min per scan for an area of 100 × 100 µm2 and are accompanied by morphological information, supplied by a fast AFM, of the same area. Hence, a complete reactivity overview is obtained within several hours with only a monolayer of reactant. To demonstrate the working principle of this method, a SERS substrate containing the alignment pattern and silver nanoparticle aggregates as catalytic sites is prepared to study the photo-catalytic reduction of p-nitrothiophenol ( p-NTP).

Understanding Ultrafast Dynamics of Conformation Specific Photo-Excitation: A Femtosecond Transient Absorption and Ultrafast Raman Loss Study.

Excited state ultrafast conformational reorganization is recognized as an important phenomenon that facilitates light-induced functions of many molecular systems. This report describes the femtosecond and picosecond conformational relaxation dynamics of middle-ring and terminal ring twisted conformers of the acetylene π-conjugated system bis(phenylethynyl)benzene, a model system for molecular wires. Through excitation wavelength dependent, femtosecond-transient absorption measurements, we found that the middle-ring and terminal ring twisted conformers relax at femtosecond (400-600 fs) and picosecond (20-24 ps) time scales, respectively. Actinic pumping into the red flank of the absorption spectrum leads to excitation of primarily planar conformers, and results in very different excited state dynamics. In addition, ultrafast Raman loss spectroscopic studies revealed the vibrational mode dependent relaxation dynamics for different excitation wavelengths. To corroborate our experimental findings, DFT and time-dependent DFT calculations were carried out. The Franck-Condon simulation indicated that the vibronic structure observed in the electronic absorption and the fluorescence spectra are due to progressions and combinations of several vibrational modes corresponding to the phenyl ring and the acetylenic groups. Furthermore, the middle ring torsional rotation matches the room-temperature electronic absorption, in stark contrast to the terminal ring torsional rotation. Finally, we show that the middle-ring twisted conformer undergoes femtosecond torsional planarization dynamic, whereas the terminal rings relax on a few tens of picosecond time scale.