Real-time stimulated Raman spectroscopy with a non-collinear optical parametric oscillator.

Ultrafast detection of microplastic particles is becoming a vital problem, as these particles are found in water sources worldwide. Ideally, a live analysis in flow is desirable to directly monitor the water quality for contaminations. Therefore, coherent Raman spectroscopy techniques require fast and broadband tunable lasers to address all relevant spectral regions of the investigated samples. In our work, we combine a high power non-collinear optical parametric oscillator with a real-time stimulated Raman scattering spectroscopy setup. The light source is continously tunable from 700 nm to 1030 nm in less than 10 ms, delivering an average output power of more than 500 mW with sub-ps pulses. We show the immediate observation of mixing processes and the detection of microplastic particles in water solution with a spectral window of more than 2000 cm-1.

Calcium carbonate deposits and microbial assemblages on microplastics in oligotrophic freshwaters.

Microplastics are solid polymer particles with a wide variety of surface properties, found in most waterbodies, and known as carriers of distinct microbial communities affecting the fate of the particles in the environment. Little is known about the formation of mineral deposits on microplastics and how these deposits connect to microbial assemblages and affect the physicochemical properties of the particles. In addition, most of the available research on this topic is based on large microplastics with sizes between 100 µm and up to 5 mm, rather than the small microplastics often found in drinking water sources. To narrow this gap in our understanding of environmental effects on small microplastics, two types of small microplastics made of two distinct polymers, poly(methyl methacrylate) (PMMA) and poly(tetrafluoroethylene) (PTFE) with sizes ranging from 15 to 150 µm, were incubated for six months in unprocessed and processed drinking water with increasing ionic concentration to allow for the formation of mineral deposits and microbial assemblages. Spatially resolved analysis with fluorescent in situ hybridization and confocal Raman microscopic imaging revealed deposits of calcium carbonates and scattered microbial assemblages on all microplastics, with structure, extend, and microbial association with the carbonates depending on the respective microplastic. Notably, PTFE floatation was overcome after three months in unprocessed drinking water but remained unchanged in processed drinking water, whereas PMMA appeared unaffected, indicating that the fate of microplastics in the environment may depend on polymer type and the encountered aquatic conditions forming mineral and microbial attachments to the particle surface.

Small Sample Stress: Probing Oxygen-Deprived Ammonia-Oxidizing Bacteria with Raman Spectroscopy In Vivo.

The stress response of ammonia-oxidizing bacteria (AOB) to oxygen deprivation limits AOB growth and leads to different nitrification pathways that cause the release of greenhouse gases. Measuring the stress response of AOB has proven to be a challenge due to the low growth rates of stressed AOB, making the sample volumes required to monitor the internal stress response of AOB prohibitive to repeated analysis. In a proof-of-concept study, confocal Raman microscopy with excitation resonant to the heme c moiety of cytochrome c was used to compare the cytochrome c content and activity of stressed and unstressed Nitrosomonas europaea (Nm 50), Nitrosomonas eutropha (Nm 57), Nitrosospira briensis (Nsp 10), and Nitrosospira sp. (Nsp 02) in vivo. Each analysis required no more than 1000 individual cells per sampling; thus, the monitoring of cultures with low cell concentrations was possible. The identified spectral marker delivered reproducible results within the signal-to-noise ratio of the underlying Raman spectra. Cytochrome c content was found to be elevated in oxygen-deprived and previously oxygen-deprived samples. In addition, cells with predominantly ferrous cytochrome c content were found in deprived Nitrosomonas eutropha and Nitrosospira samples, which may be indicative of ongoing electron storage at the time of measurement.

pH-Dependent Conformational Changes of KcsA Tetramer and Monomer Probed by Raman Spectroscopy.

KcsA is a tetrameric potassium channel formed out of four identical monomeric subunits used as a standard model for selective potassium transport and pH-dependent gating. Large conformational changes are reported for tetramer and monomer upon gating, and the response of the monomer being controversial with the two major studies partially contradicting each other. KcsA was analyzed as functional tetramers embedded in liposomes and as monomer subunits with confocal Raman microscopy under physiological conditions for the active and the closed channel state, using 532 nm excitation to avoid introducing conformational changes during the measurement. Channel function was confirmed using liposome flux assay. While the classic fingerprint region below 1800 rel. cm-1 in the Raman spectrum of the tetramer was unaffected, the CH-stretching region between 2800 and 3200 rel. cm-1 was found to be strongly affected by the conformation. No pH-dependency was observed in the Raman spectra of the monomer subunits, which closely resembled the Raman spectrum of the tetramer in its active conformation, indicating that the open conformation of the monomer and not the closed conformation as postulated may equal the relaxed state of the molecule.

Microplastics Detection in Streaming Tap Water with Raman Spectroscopy.

Microplastic particles have been found in drinking water sources worldwide and, thus, also in our food and beverages. Especially small microplastics, with sizes of 1 mm and less, cannot be identified reliably without spectroscopic means such as Fourier transform infrared spectroscopy (FTIR) or Raman spectroscopy, usually applied to the particles extracted from the samples. However, for drinking and tap water, with its comparatively low biological loads, direct observation may be possible and allows a point-of-entry monitoring for beverages and food to ensure uncontaminated drinking water is being used. In a proof of concept, we apply Raman spectroscopy to observe individual microplastic particles in tap water with added particulate and fluorescent contaminants streaming with 1 L/h through a custom-made flow cell. We evaluated several tubing materials for compatibility with microplastic suspensions containing three different polymers widely found in microplastic surveys worldwide. The experiment promises the monitoring of streaming tap water and even clear surface waters for microplastics smaller than 0.1 mm.

Simulation of Raman scattering including detector parameters and sampling volume.

Raman spectroscopy can be employed to measure the chemical composition of a sample, which can in turn be used to extract biological information. The aim of this paper is to introduce an efficient simulation technique for Raman spectroscopy in turbid (scattering) media taking into account relevant detector parameters and the sampling volume. We simulate the process of photon motion in turbid media by means of the Monte Carlo (MC) method. The numerical simulation of Raman scattering consists of two stages: calculation of the photon fluence at each point of the medium and subsequent generation of the corresponding amount of Raman photons at each point. The developed model allows simulation of both confocal and optical fiber probe Raman setups. In more detail, the model efficiently simulates Raman signals for different single and multi-layer phantoms and geometries, including focused and collimated (i.e., the fiber-based case) excitation laser beams as well as different values for the numerical aperture and the excitation beam radius. In the future, our results offer the potential to improve the design of Raman systems for in vivo applications in biomedical research.

Heat and Bleach: A Cost-Efficient Method for Extracting Microplastics from Return Activated Sludge.

The extraction of plastic microparticles, so-called microplastics, from sludge is a challenging task due to the complex, highly organic material often interspersed with other benign microparticles. The current procedures for microplastic extraction from sludge are time consuming and require expensive reagents for density separation as well as large volumes of oxidizing agents for organic removal, often resulting in tiny sample sizes and thus a disproportional risk of sample bias. In this work, we present an improved extraction method tested on return activated sludge (RAS). The treatment of 100 ml of RAS requires only 6% hydrogen peroxide (H2O2) for bleaching at 70 °C, followed by density separation with sodium nitrate/sodium thiosulfate (SNT) solution, and is completed within 24 h. Extracted particles of all sizes were chemically analyzed with confocal Raman microscopy. An extraction efficiency of 78 ± 8% for plastic particle sizes 20 µm and up was confirmed in a recovery experiment. However, glass shards with a diameter of less than 20 µm remained in the sample despite the density of glass exceeding the density of the separating SNT solution by 1.1 g/cm3. This indicates that density separation may be unreliable for particle sizes in the lower micrometer range.

Confocal Raman microscopy and fluorescent in situ hybridization - A complementary approach for biofilm analysis.

We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples.

Temperature-sensitive gating of hCx26: high-resolution Raman spectroscopy sheds light on conformational changes.

The temperature-sensitive gating of human Connexin 26 (hCx26) was analyzed with confocal Raman microscopy. High-resolution Raman spectra covering the spectral range between 400 and 1500 rel. cm(-1) with a spectral resolution of 1 cm(-1) were fully annotated, revealing notable differences between the spectrum recorded from solubilized hCx26 in Ca(2+)-buffered POPC at 10°C and any other set of protein conditions (temperature, Ca(2+) presence, POPC presence). Spectral components originating from specific amino acids show that the TM1/EL1 parahelix and probably the TM4 trans-membrane helix and the plug domain are involved in the gating process responsible for fully closing the hemichannel.

Of microparticles and bacteria identification--(resonance) Raman micro-spectroscopy as a tool for biofilm analysis.

Confocal resonance Raman microscopy is a powerful tool for the non-invasive analysis of complex biological aggregates without preparation and prior knowledge of the samples. We present the capabilities of confocal resonance Raman microscopy with a spatial resolution of 350 nm2×2.0 µm and excitation times of 1 s and less per recorded spectrum. Granules sampled from two sequencing batch reactors (SBR) for anaerobic ammonium oxidization (anammox) were regularly mapped in vivo for three months after SBR startup. Uncultured microorganisms and mineral particles were tracked throughout operation and identified in situ by their (resonance) Raman spectra. Co-existing microcolonies of Nitrosomonae formed the outer layer of anammox granules. Polymorph TiO2 microparticles were found embedded in the outer layer of granules overgrown with purple bacteria, indicating bacterial response to the variant toxicity of the mineral phase.

Effects of ethanol, formaldehyde, and gentle heat fixation in confocal resonance Raman microscopy of purple nonsulfur bacteria.

Resonance Raman microscopy is well suited to examine living bacterial samples without further preparation. Therefore, comparatively little thought has been given to its compatibility with common fixation methods. However, fixation of cell samples is a very important tool in the microbiological sciences, allowing the preservation of samples in a specific condition for further examination, future measurements, transport, or later reference. We examined the effects of three common fixatives-ethanol, formaldehyde solution, and gentle heat--on the resonant Raman spectrum of three generic bacteria species, Rhodobacter sphaeroides DSM 158(T), Rhodopseudomonas palustris DSM 123(T), and Rhodospirillum rubrum DSM 467(T), holding carotenoid- and heme-chromophores in confocal Raman microscopy. In addition, we analyzed the effect of poly-L-lysine coating of microscope slides, widely used for mounting biological and medical samples, on subsequent confocal Raman measurements of native and fixed samples. The results indicate that ethanol is preferable to formaldehyde as fixative if applied for less than 24 h, whereas heat fixation has a strong, detrimental effect on the resonant Raman spectrum of bacteria. Formaldehyde fixation excels at fixation times above 24 h, but causes an overall reduction in signal intensity. Poly-L-lysine coating has no discernable effect on the Raman spectra of samples fixed with ethanol or heat, but it further decreases the signal intensity, especially at higher wavenumbers, in the spectra of samples fixed with formaldehyde.