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- ItemFusion of MALDI Spectrometric Imaging and Raman Spectroscopic Data for the Analysis of Biological Samples(Lausanne : Frontiers Media, 2018) Ryabchykov, Oleg; Popp, Jürgen; Bocklitz, Thomas W.Despite of a large number of imaging techniques for the characterization of biological samples, no universal one has been reported yet. In this work, a data fusion approach was investigated for combining Raman spectroscopic data with matrix-assisted laser desorption/ionization (MALDI) mass spectrometric data. It betters the image analysis of biological samples because Raman and MALDI information can be complementary to each other. While MALDI spectrometry yields detailed information regarding the lipid content, Raman spectroscopy provides valuable information about the overall chemical composition of the sample. The combination of Raman spectroscopic and MALDI spectrometric imaging data helps distinguishing different regions within the sample with a higher precision than would be possible by using either technique. We demonstrate that a data weighting step within the data fusion is necessary to reveal additional spectral features. The selected weighting approach was evaluated by examining the proportions of variance within the data explained by the first principal components of a principal component analysis (PCA) and visualizing the PCA results for each data type and combined data. In summary, the presented data fusion approach provides a concrete guideline on how to combine Raman spectroscopic and MALDI spectrometric imaging data for biological analysis.
- ItemOnline investigation of respiratory quotients in Pinus sylvestris and Picea abies during drought and shading by means of cavity-enhanced Raman multi-gas spectrometry(Cambridge : Soc., 2015) Hanf, Stefan; Fischer, Sarah; Hartmann, Henrik; Keiner, Robert; Trumbore, Susan; Popp, Jürgen; Frosch, TorstenPhotosynthesis and respiration are major components of the plant carbon balance. During stress, like drought, carbohydrate supply from photosynthesis is reduced and the Krebs cycle respiration must be fueled with other stored carbon compounds. However, the dynamics of storage use are still unknown. The respiratory quotient (RQ, CO2 released per O2 consumed during respiration) is an excellent indicator of the nature of the respiration substrate. In plant science, however, online RQ measurements have been challenging or even impossible so far due to very small gas exchange fluxes during respiration. Here we apply cavity-enhanced multi-gas Raman spectrometry (CERS) for online in situ RQ measurements in drought-tolerant pine (Pinus sylvestris [L.]) and drought-intolerant spruce (Picea abies [L. H. Karst]). Two different treatments, drought and shading, were applied to reduce photosynthesis and force dependency on stored substrates. Changes in respiration rates and RQ values were continuously monitored over periods of several days with low levels of variance. The results show that both species switched from COH-dominated respiration (RQ = 1.0) to a mixture of substrates during shading (RQ = 0.77–0.81), while during drought only pine did so (RQ = 0.75). The gas phase measurements were complemented by concentration measurements of non-structural carbohydrates and lipids. These first results suggest a physiological explanation for greater drought tolerance in pine. CERS was proven as powerful technique for non-consumptive and precise real-time monitoring of respiration rates and respirational quotients for the investigation of plant metabolism under drought stress conditions that are predicted to increase with future climate change.
- ItemMicrobial respiration and natural attenuation of benzene contaminated soils investigated by cavity enhanced Raman multi-gas spectroscopy(Cambridge : Soc., 2015) Jochum, Tobias; Michalzik, Beate; Bachmann, Anne; Popp, Jürgen; Frosch, TorstenSoil and groundwater contamination with benzene can cause serious environmental damage. However, many soil microorganisms are capable to adapt and are known to strongly control the fate of organic contamination. Innovative cavity enhanced Raman multi-gas spectroscopy (CERS) was applied to investigate the short-term response of the soil micro-flora to sudden surface contamination with benzene regarding the temporal variations of gas products and their exchange rates with the adjacent atmosphere. 13C-labeled benzene was spiked on a silty-loamy soil column in order to track and separate the changes in heterotrophic soil respiration – involving 12CO2 and O2 – from the natural attenuation process of benzene degradation to ultimately form 13CO2. The respiratory quotient (RQ) decreased from a value 0.98 to 0.46 directly after the spiking and increased again within 33 hours to a value of 0.72. This coincided with the maximum 13CO2 concentration rate (0.63 μmol m−2 s−1), indicating the highest benzene degradation at 33 hours after the spiking event. The diffusion of benzene in the headspace and the biodegradation into 13CO2 were simultaneously monitored and 12 days after the benzene spiking no measurable degradation was detected anymore. The RQ finally returned to a value of 0.96 demonstrating the reestablished aerobic respiration.
- ItemFLIm and raman spectroscopy for investigating biochemical changes of bovine pericardium upon genipin cross-linking(Basel : MDPI, 2020) Shaik, Tanveer Ahmed; Alfonso-Garcia, Alba; Richter, Martin; Korinth, Florian; Krafft, Christoph; Marcu, Laura; Popp, JürgenBiomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm-1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemRevealing the Chemical Composition of Birch Pollen Grains by Raman Spectroscopic Imaging(Basel : Molecular Diversity Preservation International (MDPI), 2022) Stiebing, Clara; Post, Nele; Schindler, Claudia; Göhrig, Bianca; Lux, Harald; Popp, Jürgen; Heutelbeck, Astrid; Schie, Iwan W.The investigation of the biochemical composition of pollen grains is of the utmost interest for several environmental aspects, such as their allergenic potential and their changes in growth conditions due to climatic factors. In order to fully understand the composition of pollen grains, not only is an in-depth analysis of their molecular components necessary but also spatial information of, e.g., the thickness of the outer shell, should be recorded. However, there is a lack of studies using molecular imaging methods for a spatially resolved biochemical composition on a single-grain level. In this study, Raman spectroscopy was implemented as an analytical tool to investigate birch pollen by imaging single pollen grains and analyzing their spectral profiles. The imaging modality allowed us to reveal the layered structure of pollen grains based on the biochemical information of the recorded Raman spectra. Seven different birch pollen species collected at two different locations in Germany were investigated and compared. Using chemometric algorithms such as hierarchical cluster analysis and multiple-curve resolution, several components of the grain wall, such as sporopollenin, as well as the inner core presenting high starch concentrations, were identified and quantified. Differences in the concentrations of, e.g., sporopollenin, lipids and proteins in the pollen species at the two different collection sites were found, and are discussed in connection with germination and other growth processes.
- ItemAssessment of shifted excitation Raman difference spectroscopy in highly fluorescent biological samples(Cambridge : Soc., 2021) Korinth, Florian; Shaik, Tanveer Ahmed; Popp, Jürgen; Krafft, ChristophShifted excitation Raman difference spectroscopy (SERDS) can be used as an instrumental baseline correction technique to retrieve Raman bands in highly fluorescent samples. Genipin (GE) cross-linked equine pericardium (EP) was used as a model system since a blue pigment is formed upon cross-linking, which results in a strong fluorescent background in the Raman spectra. EP was cross-linked with 0.25% GE solution for 0.5 h, 2 h, 4 h, 6 h, 12 h, and 24 h, and compared with corresponding untreated EP. Raman spectra were collected with three different excitation wavelengths. For the assessment of the SERDS technique, the preprocessed SERDS spectra of two excitation wavelengths (784 nm-786 nm) were compared with the mathematical baseline-corrected Raman spectra at 785 nm excitation using extended multiplicative signal correction, rubberband, the sensitive nonlinear iterative peak and polynomial fitting algorithms. Whereas each baseline correction gave poor quality spectra beyond 6 h GE crosslinking with wave-like artefacts, the SERDS technique resulted in difference spectra, that gave superior reconstructed spectra with clear collagen and resonance enhanced GE pigment bands with lower standard deviation. Key for this progress was an advanced difference optimization approach that is described here. Furthermore, the results of the SERDS technique were independent of the intensity calibration because the system transfer response was compensated by calculating the difference spectrum. We conclude that this SERDS strategy can be transferred to Raman studies on biological and non-biological samples with a strong fluorescence background at 785 nm and also shorter excitation wavelengths which benefit from more intense scattering intensities and higher quantum efficiencies of CCD detectors. This journal is
- ItemNovel Biobased Self-Healing Ionomers Derived from Itaconic Acid Derivates(Weinheim : Wiley-VCH, 2021) Meurer, Josefine; Hniopek, Julian; Dahlke, Jan; Schmitt, Michael; Popp, Jürgen; Zechel, Stefan; Hager, Martin D.This article presents novel biobased ionomers featuring self-healing abilities. These smart materials are synthesized from itaconic acid derivates. Large quantities of itaconic acid can be produced from diverse biomass like corn, rice, and others. This study presents a comprehensive investigation of their thermal and mechanical properties via differential scanning calorimetry (DSC), thermo gravimetric analysis (TGA), and FT-Raman and FT-IR measurements as well as dynamic mechanic analysis. Within all these measurements, different kinds of structure-property relationships could be derived from these measurements. For example, the proportion of ionic groups enormously influences the self-healing efficiency. The investigation of the self-healing abilities reveals healing efficiencies up to 99% in 2 h at 90 °C for the itaconic acid based ionomer with the lowest ionic content. © 2020 The Authors. Macromolecular Rapid Communications published by Wiley-VCH GmbH
- ItemLinear and non-linear optical imaging of cancer cells with silicon nanoparticles(Basel : Molecular Diversity Preservation International (MDPI), 2016) Tolstik, Elen; Osminkina, Liubov A.; Akimov, Denis; Gongalsky, Maksim B.; Kudryavtsev, Andrew A.; Timoshenko, Victor Yu.; Heintzmann, Rainer; Sivakov, Vladimir; Popp, JürgenNew approaches for visualisation of silicon nanoparticles (SiNPs) in cancer cells are realised by means of the linear and nonlinear optics in vitro. Aqueous colloidal solutions of SiNPs with sizes of about 10–40 nm obtained by ultrasound grinding of silicon nanowires were introduced into breast cancer cells (MCF-7 cell line). Further, the time-varying nanoparticles enclosed in cell structures were visualised by high-resolution structured illumination microscopy (HR-SIM) and micro-Raman spectroscopy. Additionally, the nonlinear optical methods of two-photon excited fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS) with infrared laser excitation were applied to study the localisation of SiNPs in cells. Advantages of the nonlinear methods, such as rapid imaging, which prevents cells from overheating and larger penetration depth compared to the single-photon excited HR-SIM, are discussed. The obtained results reveal new perspectives of the multimodal visualisation and precise detection of the uptake of biodegradable non-toxic SiNPs by cancer cells and they are discussed in view of future applications for the optical diagnostics of cancer tumours.
- ItemA Machine Learning-Based Raman Spectroscopic Assay for the Identification of Burkholderia mallei and Related Species(Basel : MDPI, 2019) Silge, Anja; Moawad, Amira A.; Bocklitz, Thomas; Fischer, Katja; Rösch, Petra; Roesler, Uwe; Elschner, Mandy C.; Popp, Jürgen; Neubauer, HeinrichBurkholderia (B.) mallei, the causative agent of glanders, and B. pseudomallei, the causative agent of melioidosis in humans and animals, are genetically closely related. The high infectious potential of both organisms, their serological cross-reactivity, and similar clinical symptoms in human and animals make the differentiation from each other and other Burkholderia species challenging. The increased resistance against many antibiotics implies the need for fast and robust identification methods. The use of Raman microspectroscopy in microbial diagnostic has the potential for rapid and reliable identification. Single bacterial cells are directly probed and a broad range of phenotypic information is recorded, which is subsequently analyzed by machine learning methods. Burkholderia were handled under biosafety level 1 (BSL 1) conditions after heat inactivation. The clusters of the spectral phenotypes and the diagnostic relevance of the Burkholderia spp. were considered for an advanced hierarchical machine learning approach. The strain panel for training involved 12 B. mallei, 13 B. pseudomallei and 11 other Burkholderia spp. type strains. The combination of top- and sub-level classifier identified the mallei-complex with high sensitivities (>95%). The reliable identification of unknown B. mallei and B. pseudomallei strains highlighted the robustness of the machine learning-based Raman spectroscopic assay. © 2019 by the authors
- ItemVibrational Spectroscopic Investigation of Blood Plasma and Serum by Drop Coating Deposition for Clinical Application(Basel : Molecular Diversity Preservation International (MDPI), 2021) Huang, Jing; Ali, Nairveen; Quansah, Elsie; Guo, Shuxia; Noutsias, Michel; Meyer-Zedler, Tobias; Bocklitz, Thomas; Popp, Jürgen; Neugebauer, Ute; Ramoji, AnuradhaIn recent decades, vibrational spectroscopic methods such as Raman and FT-IR spectroscopy are widely applied to investigate plasma and serum samples. These methods are combined with drop coating deposition techniques to pre-concentrate the biomolecules in the dried droplet to improve the detected vibrational signal. However, most often encountered challenge is the inhomogeneous redistribution of biomolecules due to the coffee-ring effect. In this study, the variation in biomolecule distribution within the dried-sample droplet has been investigated using Raman and FT-IR spectroscopy and fluorescence lifetime imaging method. The plasma-sample from healthy donors were investigated to show the spectral differences between the inner and outer-ring region of the dried-sample droplet. Further, the preferred location of deposition of the most abundant protein albumin in the blood during the drying process of the plasma has been illustrated by using deuterated albumin. Subsequently, two patients with different cardiac-related diseases were investigated exemplarily to illustrate the variation in the pattern of plasma and serum biomolecule distribution during the drying process and its impact on patient-stratification. The study shows that a uniform sampling position of the droplet, both at the inner and the outer ring, is necessary for thorough clinical characterization of the patient’s plasma and serum sample using vibrational spectroscopy.