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- ItemUltra-compact tunable fiber laser for coherent anti-Stokes Raman imaging(Chichester [u.a.] : Wiley, 2021) Gottschall, Thomas; Meyer-Zedler, Tobias; Schmitt, Michael; Huber, Robert; Popp, Juergen; Tünnermann, Andreas; Limpert, JensThis work describes the construction of an ultra-compact narrowband fiber laser source for coherent anti-Stokes Raman scattering microscopy of Raman tags, that is, for addressing Raman resonances of deuterated molecules and alkyne tags in the spectral range from 2080 to 2220 cm−1. A narrowband and fast electronically tunable cw seed source based on a semiconductor optical amplifier (SOA) emitting around 1335 nm has been employed to seed four-wave mixing (FWM) in an endlessly single mode fiber (ESM) pumped by a ps pulse duration Yb-fiber laser. A conversion efficiency of 50% is demonstrated. This compact fiber optical parametric amplifier (FOPA) has been used to perform coherent anti-Stokes Raman imaging experiments of crystalline deuterated palmitic acid.
- ItemFLIM data analysis based on Laguerre polynomial decomposition and machine-learning(Bellingham, Wash. : SPIE, 2021) Guo, Shuxia; Silge, Anja; Bae, Hyeonsoo; Tolstik, Tatiana; Meyer, Tobias; Matziolis, Georg; Schmitt, Michael; Popp, Jürgen; Bocklitz, ThomasSignificance: The potential of fluorescence lifetime imaging microscopy (FLIM) is recently being recognized, especially in biological studies. However, FLIM does not directly measure the lifetimes, rather it records the fluorescence decay traces. The lifetimes and/or abundances have to be estimated from these traces during the phase of data processing. To precisely estimate these parameters is challenging and requires a well-designed computer program. Conventionally employed methods, which are based on curve fitting, are computationally expensive and limited in performance especially for highly noisy FLIM data. The graphical analysis, while free of fit, requires calibration samples for a quantitative analysis. Aim: We propose to extract the lifetimes and abundances directly from the decay traces through machine learning (ML). Approach: The ML-based approach was verified with simulated testing data in which the lifetimes and abundances were known exactly. Thereafter, we compared its performance with the commercial software SPCImage based on datasets measured from biological samples on a time-correlated single photon counting system. We reconstructed the decay traces using the lifetime and abundance values estimated by ML and SPCImage methods and utilized the root-mean-squared-error (RMSE) as marker. Results: The RMSE, which represents the difference between the reconstructed and measured decay traces, was observed to be lower for ML than for SPCImage. In addition, we could demonstrate with a three-component analysis the high potential and flexibility of the ML method to deal with more than two lifetime components.
- ItemA polyyne toxin produced by an antagonistic bacterium blinds and lyses a Chlamydomonad alga(Washington, DC : National Acad. of Sciences, 2021) Hotter, Vivien; Zopf, David; Kim, Hak Joong; Silge, Anja; Schmitt, Michael; Aiyar, Prasad; Fleck, Johanna; Matthäus, Christian; Hniopek, Julian; Yan, Qing; Loper, Joyce; Sasso, Severin; Hertweck, Christian; Popp, Jürgen; Mittag, MariaAlgae are key contributors to global carbon fixation and form the basis of many food webs. In nature, their growth is often supported or suppressed by microorganisms. The bacterium Pseudomonas protegens Pf-5 arrests the growth of the green unicellular alga Chlamydomonas reinhardtii, deflagellates the alga by the cyclic lipopeptide orfamide A, and alters its morphology [P. Aiyar et al., Nat. Commun. 8, 1756 (2017)]. Using a combination of Raman microspectroscopy, genome mining, and mutational analysis, we discovered a polyyne toxin, protegencin, which is secreted by P. protegens, penetrates the algal cells, and causes destruction of the carotenoids of their primitive visual system, the eyespot. Together with secreted orfamide A, protegencin thus prevents the phototactic behavior of C. reinhardtii. A mutant of P. protegens deficient in protegencin production does not affect growth or eyespot carotenoids of C. reinhardtii. Protegencin acts in a direct and destructive way by lysing and killing the algal cells. The toxic effect of protegencin is also observed in an eyeless mutant and with the colony-forming Chlorophyte alga Gonium pectorale. These data reveal a two-pronged molecular strategy involving a cyclic lipopeptide and a conjugated tetrayne used by bacteria to attack select Chlamydomonad algae. In conjunction with the bloom-forming activity of several chlorophytes and the presence of the protegencin gene cluster in over 50 different Pseudomonas genomes [A. J. Mullins et al., bioRxiv [Preprint] (2021). https://www.biorxiv.org/content/10.1101/2021.03.05.433886v1 (Accessed 17 April 2021)], these data are highly relevant to ecological interactions between Chlorophyte algae and Pseudomonadales bacteria.
- 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
- ItemIntestinal epithelial barrier integrity investigated by label-free techniques in ulcerative colitis patients([London] : Macmillan Publishers Limited, part of Springer Nature, 2023) Quansah, Elsie; Gardey, Elena; Ramoji, Anuradha; Meyer-Zedler, Tobias; Goehrig, Bianca; Heutelbeck, Astrid; Hoeppener, Stephanie; Schmitt, Michael; Waldner, Maximillian; Stallmach, Andreas; Popp, JürgenThe intestinal epithelial barrier, among other compartments such as the mucosal immune system, contributes to the maintenance of intestinal homeostasis. Therefore, any disturbance within the epithelial layer could lead to intestinal permeability and promote mucosal inflammation. Considering that disintegration of the intestinal epithelial barrier is a key element in the etiology of ulcerative colitis, further assessment of barrier integrity could contribute to a better understanding of the role of epithelial barrier defects in ulcerative colitis (UC), one major form of chronic inflammatory bowel disease. Herein, we employ fast, non-destructive, and label-free non-linear methods, namely coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and two-photon fluorescence lifetime imaging (2P-FLIM), to assess the morpho-chemical contributions leading to the dysfunction of the epithelial barrier. For the first time, the formation of epithelial barrier gaps was directly visualized, without sophisticated data analysis procedures, by the 3D analysis of the colonic mucosa from severely inflamed UC patients. The results were compared with histopathological and immunofluorescence images and validated using transmission electron microscopy (TEM) to indicate structural alterations of the apical junction complex as the underlying cause for the formation of the epithelial barrier gaps. Our findings suggest the potential advantage of non-linear multimodal imaging is to give precise, detailed, and direct visualization of the epithelial barrier in the gastrointestinal tract, which can be combined with a fiber probe for future endomicroscopy measurements during real-time in vivo imaging.
- 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
- ItemIn vivo coherent anti-Stokes Raman scattering microscopy reveals vitamin A distribution in the liver(Weinheim : Wiley-VCH-Verl., 2021) Rodewald, Marko; Bae, Hyeonsoo; Huschke, Sophie; Meyer-Zedler, Tobias; Schmitt, Michael; Press, Adrian Tibor; Schubert, Stephanie; Bauer, Michael; Popp, JuergenHere we present a microscope setup for coherent anti-Stokes Raman scattering (CARS) imaging, devised to specifically address the challenges of in vivo experiments. We exemplify its capabilities by demonstrating how CARS microscopy can be used to identify vitamin A (VA) accumulations in the liver of a living mouse, marking the positions of hepatic stellate cells (HSCs). HSCs are the main source of extracellular matrix protein after hepatic injury and are therefore the main target of novel nanomedical strategies in the development of a treatment for liver fibrosis. Their role in the VA metabolism makes them an ideal target for a CARS-based approach as they store most of the body's VA, a class of compounds sharing a retinyl group as a structural motive, a moiety that is well known for its exceptionally high Raman cross section of the C=C stretching vibration of the conjugated backbone.
- ItemLabel-free multimodal imaging of infected Galleria mellonella larvae([London] : Macmillan Publishers Limited, part of Springer Nature, 2022) Quansah, Elsie; Ramoji, Anuradha; Thieme, Lara; Mirza, Kamran; Goering, Bianca; Makarewicz, Oliwia; Heutelbeck, Astrid; Meyer-Zedler, Tobias; Pletz, Mathias W.; Schmitt, Michael; Popp, JürgenNon-linear imaging modalities have enabled us to obtain unique morpho-chemical insights into the tissue architecture of various biological model organisms in a label-free manner. However, these imaging techniques have so far not been applied to analyze the Galleria mellonella infection model. This study utilizes for the first time the strength of multimodal imaging techniques to explore infection-related changes in the Galleria mellonella larvae due to massive E. faecalis bacterial infection. Multimodal imaging techniques such as fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) were implemented in conjunction with histological HE images to analyze infection-associated tissue damage. The changes in the larvae in response to the infection, such as melanization, vacuolization, nodule formation, and hemocyte infiltration as a defense mechanism of insects against microbial pathogens, were visualized after Enterococcus faecalis was administered. Furthermore, multimodal imaging served for the analysis of implant-associated biofilm infections by visualizing biofilm adherence on medical stainless steel and ePTFE implants within the larvae. Our results suggest that infection-related changes as well as the integrity of the tissue of G. mellonella larvae can be studied with high morphological and chemical contrast in a label-free manner.