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- ItemIn-vivo Raman spectroscopy: from basics to applications(Bellingham, Wash. : SPIE, 2018) Cordero, Eliana; Latka, Ines; Matthäus, Christian; Schie, Iwan W.; Popp, JürgenFor more than two decades, Raman spectroscopy has found widespread use in biological and medical applications. The instrumentation and the statistical evaluation procedures have matured, enabling the lengthy transition from ex-vivo demonstration to in-vivo examinations. This transition goes hand-in-hand with many technological developments and tightly bound requirements for a successful implementation in a clinical environment, which are often difficult to assess for novice scientists in the field. This review outlines the required instrumentation and instrumentation parameters, designs, and developments of fiber optic probes for the in-vivo applications in a clinical setting. It aims at providing an overview of contemporary technology and clinical trials and attempts to identify future developments necessary to bring the emerging technology to the clinical end users. A comprehensive overview of in-vivo applications of fiber optic Raman probes to characterize different tissue and disease types is also given.
- ItemNew perspectives for viability studies with high-content analysis Raman spectroscopy (HCA-RS)(Berlin : Nature Publishing, 2019) Mondol, Abdullah S.; Töpfer, Natalie; Rüger, Jan; Neugebauer, Ute; Popp, Jürgen; Schie, Iwan W.Raman spectroscopy has been widely used in clinical and molecular biological studies, providing high chemical specificity without the necessity of labels and with little-to-no sample preparation. However, currently performed Raman-based studies of eukaryotic cells are still very laborious and time-consuming, resulting in a low number of sampled cells and questionable statistical validations. Furthermore, the approach requires a trained specialist to perform and analyze the experiments, rendering the method less attractive for most laboratories. In this work, we present a new high-content analysis Raman spectroscopy (HCA-RS) platform that overcomes the current challenges of conventional Raman spectroscopy implementations. HCA-RS allows sampling of a large number of cells under different physiological conditions without any user interaction. The performance of the approach is successfully demonstrated by the development of a Raman-based cell viability assay, i.e., the effect of doxorubicin concentration on monocytic THP-1 cells. A statistical model, principal component analysis combined with support vector machine (PCA-SVM), was found to successfully predict the percentage of viable cells in a mixed population and is in good agreement to results obtained by a standard cell viability assay. This study demonstrates the potential of Raman spectroscopy as a standard high-throughput tool for clinical and biological applications.
- ItemModified PCA and PLS: Towards a better classification in Raman spectroscopy–based biological applications(New York, NY : Wiley Interscience, 2020) Guo, Shuxia; Rösch, Petra; Popp, Jürgen; Bocklitz, ThomasRaman spectra of biological samples often exhibit variations originating from changes of spectrometers, measurement conditions, and cultivation conditions. Such unwanted variations make a classification extremely challenging, especially if they are more significant compared with the differences between groups to be separated. A classifier is prone to such unwanted variations (ie, intragroup variations) and can fail to learn the patterns that can help separate different groups (ie, intergroup differences). This often leads to a poor generalization performance and a degraded transferability of the trained model. A natural solution is to separate the intragroup variations from the intergroup differences and build the classifier based on merely the latter information, for example, by a well-designed feature extraction. This forms the idea of this contribution. Herein, we modified two commonly applied feature extraction approaches, principal component analysis (PCA) and partial least squares (PLS), in order to extract merely the features representing the intergroup differences. Both of the methods were verified with two Raman spectral datasets measured from bacterial cultures and colon tissues of mice, respectively. In comparison to ordinary PCA and PLS, the modified PCA was able to improve the prediction on the testing data that bears significant difference to the training data, while the modified PLS could help avoid overfitting and lead to a more stable classification. © 2019 The Authors. Journal of Chemometrics published by John Wiley & Sons Ltd
- ItemComparison of Different Label-Free Raman Spectroscopy Approaches for the Discrimination of Clinical MRSA and MSSA Isolates(Birmingham, Ala. : ASM, 2022) Pistiki, Aikaterini; Monecke, Stefan; Shen, Haodong; Ryabchykov, Oleg; Bocklitz, Thomas W.; Rösch, Petra; Ehricht, Ralf; Popp, JürgenMethicillin-resistant Staphylococcus aureus (MRSA) is classified as one of the priority pathogens that threaten human health. Resistance detection with conventional microbiological methods takes several days, forcing physicians to administer empirical antimicrobial treatment that is not always appropriate. A need exists for a rapid, accurate, and cost-effective method that allows targeted antimicrobial therapy in limited time. In this pilot study, we investigate the efficacy of three different label-free Raman spectroscopic approaches to differentiate methicillin-resistant and -susceptible clinical isolates of S. aureus (MSSA). Single-cell analysis using 532 nm excitation was shown to be the most suitable approach since it captures information on the overall biochemical composition of the bacteria, predicting 87.5% of the strains correctly. UV resonance Raman microspectroscopy provided a balanced accuracy of 62.5% and was not sensitive enough in discriminating MRSA from MSSA. Excitation of 785 nm directly on the petri dish provided a balanced accuracy of 87.5%. However, the difference between the strains was derived from the dominant staphyloxanthin bands in the MRSA, a cell component not associated with the presence of methicillin resistance. This is the first step toward the development of label-free Raman spectroscopy for the discrimination of MRSA and MSSA using single-cell analysis with 532 nm excitation. IMPORTANCE Label-free Raman spectra capture the high chemical complexity of bacterial cells. Many different Raman approaches have been developed using different excitation wavelength and cell analysis methods. This study highlights the major importance of selecting the most suitable Raman approach, capable of providing spectral features that can be associated with the cell mechanism under investigation. It is shown that the approach of choice for differentiating MRSA from MSSA should be single-cell analysis with 532 nm excitation since it captures the difference in the overall biochemical composition. These results should be taken into consideration in future studies aiming for the development of label-free Raman spectroscopy as a clinical analytical tool for antimicrobial resistance determination.
- ItemApplication of High-Throughput Screening Raman Spectroscopy (HTS-RS) for Label-Free Identification and Molecular Characterization of Pollen(Basel : MDPI, 2019) Mondol, Abdullah S.; Patel, Milind D.; Rüger, Jan; Stiebing, Clara; Kleiber, Andreas; Henkel, Thomas; Popp, Jürgen; Schie, Iwan W.Pollen studies play a critical role in various fields of science. In the last couple of decades, replacement of manual identification of pollen by image-based methods using pollen morphological features was a great leap forward, but challenges for pollen with similar morphology remain, and additional approaches are required. Spectroscopy approaches for identification of pollen, such as Raman spectroscopy has potential benefits over traditional methods, due to the investigation of the intrinsic molecular composition of a sample. However, current Raman-based characterization of pollen is complex and time-consuming, resulting in low throughput and limiting the statistical significance of the acquired data. Previously demonstrated high-throughput screening Raman spectroscopy (HTS-RS) eliminates the complexity as well as human interaction by incorporation full automation of the data acquisition process. Here, we present a customization of HTS-RS for pollen identification, enabling sampling of a large number of pollen in comparison to other state-of-the-art Raman pollen investigations. We show that using Raman spectra we are able to provide a preliminary estimation of pollen types based on growth habits using hierarchical cluster analysis (HCA) as well as good taxonomy of 37 different Pollen using principal component analysis-support vector machine (PCA-SVM) with good accuracy even for the pollen specimens sharing similar morphological features. Our results suggest that HTS-RS platform meets the demands for automated pollen detection making it an alternative method for research concerning pollen.
- ItemCorrelation of crystal violet biofilm test results of Staphylococcus aureus clinical isolates with Raman spectroscopic read-out(Chichester [u.a.] : Wiley, 2021) Ebert, Christina; Tuchscherr, Lorena; Unger, Nancy; Pöllath, Christine; Gladigau, Frederike; Popp, Jürgen; Löffler, Bettina; Neugebauer, UteBiofilm-related infections occur quite frequently in hospital settings and require rapid diagnostic identification as they are recalcitrant to antibiotic therapy and make special treatment necessary. One of the standard microbiological in vitro tests is the crystal violet test. It indirectly determines the amount of biofilm by measuring the optical density (OD) of the crystal violet-stained biofilm matrix and cells. However, this test is quite time-consuming, as it requires bacterial cultivation up to several days. In this study, we correlate fast Raman spectroscopic read-out of clinical Staphylococcus aureus isolates from 47 patients with different disease background with their biofilm-forming characteristics. Included were low (OD < 10), medium (OD ≥ 10 and ≤20), and high (OD > 20) biofilm performers as determined by the crystal violet test. Raman spectroscopic analysis of the bacteria revealed most spectral differences between high and low biofilm performers in the fingerprint region between 750 and 1150 cm−1. Using partial least square regression (PLSR) analysis on the Raman spectra involving the three categories of biofilm formation, it was possible to obtain a slight linear correlation of the Raman spectra with the biofilm OD values. The PLSR loading coefficient highlighted spectral differences between high and low biofilm performers for Raman bands that represent nucleic acids, carbohydrates, and proteins. Our results point to a possible application of Raman spectroscopy as a fast prediction tool for biofilm formation of bacterial strains directly after isolation from the infected patient. This could help clinicians make timely and adapted therapeutic decision in future.
- 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.
- ItemCounterfeit and substandard test of the antimalarial tablet Riamet® by means of Raman hyperspectral multicomponent analysis(Basel : MDPI, 2019) Frosch, Timea; Wyrwich, Elisabeth; Yan, Di; Domes, Christian; Domes, Robert; Popp, Jürgen; Frosch, TorstenThe fight against counterfeit pharmaceuticals is a global issue of utmost importance, as failed medication results in millions of deaths every year. Particularly affected are antimalarial tablets. A very important issue is the identification of substandard tablets that do not contain the nominal amounts of the active pharmaceutical ingredient (API), and the differentiation between genuine products and products without any active ingredient or with a false active ingredient. This work presents a novel approach based on fiber-array based Raman hyperspectral imaging to qualify and quantify the antimalarial APIs lumefantrine and artemether directly and non-invasively in a tablet in a time-efficient way. The investigations were carried out with the antimalarial tablet Riamet® and self-made model tablets, which were used as examples of counterfeits and substandard. Partial least-squares regression modeling and density functional theory calculations were carried out for quantification of lumefantrine and artemether and for spectral band assignment. The most prominent differentiating vibrational signatures of the APIs were presented.
- ItemEvaluation of shifted excitation Raman difference spectroscopy and comparison to computational background correction methods applied to biochemical Raman spectra(Basel : MDPI, 2017) Cordero, Eliana; Korinth, Florian; Stiebing, Clara; Krafft, Christoph; Schie, Iwan W.; Popp, JürgenRaman spectroscopy provides label-free biochemical information from tissue samples without complicated sample preparation. The clinical capability of Raman spectroscopy has been demonstrated in a wide range of in vitro and in vivo applications. However, a challenge for in vivo applications is the simultaneous excitation of auto-fluorescence in the majority of tissues of interest, such as liver, bladder, brain, and others. Raman bands are then superimposed on a fluorescence background, which can be several orders of magnitude larger than the Raman signal. To eliminate the disturbing fluorescence background, several approaches are available. Among instrumentational methods shifted excitation Raman difference spectroscopy (SERDS) has been widely applied and studied. Similarly, computational techniques, for instance extended multiplicative scatter correction (EMSC), have also been employed to remove undesired background contributions. Here, we present a theoretical and experimental evaluation and comparison of fluorescence background removal approaches for Raman spectra based on SERDS and EMSC.
- ItemAutomated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins, cephalosporins, and fluoroquinolones using specific Raman marker bands(Weinheim : Wiley-VCH-Verl., 2020) Götz, Theresa; Dahms, Marcel; Kirchhoff, Johanna; Beleites, Claudia; Glaser, Uwe; Bohnert, Jürgen A.; Pletz, Mathias W.; Popp, Jürgen; Schlattmann, Peter; Neugebauer, UteA Raman-based, strain-independent, semi-automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch-wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third-generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug-resistant Gram-negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long-term comparability of Raman measurements. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim