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.

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.

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.

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.