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- 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.
- 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.
- ItemBladder tissue characterization using probe-based Raman spectroscopy: Evaluation of tissue heterogeneity and influence on the model prediction(Weinheim : Wiley-VCH-Verl., 2020) Cordero, Eliana; Rüger, Jan; Marti, Dominik; Mondol, Abdullah S.; Hasselager, Thomas; Mogensen, Karin; Hermann, Gregers G.; Popp, Jürgen; Schie, Iwan W.Existing approaches for early-stage bladder tumor diagnosis largely depend on invasive and time-consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real-time tumor diagnosis can enable immediate laser-based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real-time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe-based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low- and high-grade tumor. © 2019 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- ItemImaging the invisible—Bioorthogonal Raman probes for imaging of cells and tissues(Weinheim [u.a.] : Wiley-VCH, 2020) Azemtsop Matanfack, Georgette; Rüger, Jan; Stiebing, Clara; Schmitt, Michael; Popp, JürgenA revolutionary avenue for vibrational imaging with super-multiplexing capability can be seen in the recent development of Raman-active bioortogonal tags or labels. These tags and isotopic labels represent groups of chemically inert and small modifications, which can be introduced to any biomolecule of interest and then supplied to single cells or entire organisms. Recent developments in the field of spontaneous Raman spectroscopy and stimulated Raman spectroscopy in combination with targeted imaging of biomolecules within living systems are the main focus of this review. After having introduced common strategies for bioorthogonal labeling, we present applications thereof for profiling of resistance patterns in bacterial cells, investigations of pharmaceutical drug-cell interactions in eukaryotic cells and cancer diagnosis in whole tissue samples. Ultimately, this approach proves to be a flexible and robust tool for in vivo imaging on several length scales and provides comparable information as fluorescence-based imaging without the need of bulky fluorescent tags. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim