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- ItemPhotophysics of Anionic Bis(4H-imidazolato)CuI Complexes(Weinheim : Wiley-VCH, 2022) Seidler, Bianca; Tran, Jens H.; Hniopek, Julian; Traber, Philipp; Görls, Helmar; Gräfe, Stefanie; Schmitt, Michael; Popp, Jürgen; Schulz, Martin; Dietzek‐Ivanšić, BenjaminIn this paper, the photophysical behavior of four panchromatically absorbing, homoleptic bis(4H-imidazolato)CuI complexes, with a systematic variation in the electron-withdrawing properties of the imidazolate ligand, were studied by wavelength-dependent time-resolved femtosecond transient absorption spectroscopy. Excitation at 400, 480, and 630 nm populates metal-to-ligand charge transfer, intraligand charge transfer, and mixed-character singlet states. The pump wavelength-dependent transient absorption data were analyzed by a recently established 2D correlation approach. Data analysis revealed that all excitation conditions yield similar excited-state dynamics. Key to the excited-state relaxation is fast, sub-picosecond pseudo-Jahn-Teller distortion, which is accompanied by the relocalization of electron density onto a single ligand from the initially delocalized state at Franck-Condon geometry. Subsequent intersystem crossing to the triplet manifold is followed by a sub-100 ps decay to the ground state. The fast, nonradiative decay is rationalized by the low triplet-state energy as found by DFT calculations, which suggest perspective treatment at the strong coupling limit of the energy gap law.
- 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.
- 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.
- ItemComparison of bacteria in different metabolic states by micro-Raman spectroscopy(New York, NY [u.a.] : Elsevier, 2022) Shen, Haodong; Rösch, Petra; Thieme, Lara; Pletz, Mathias W.; Popp, JürgenIt was shown that several metabolic states of bacteria with various characteristics such as chemical composition participate in the formation of biofilms. To study the connections and differences among different bacterial metabolic states, five species of bacteria in exponential phase, stationary phase and biofilm have been compared and investigated by micro-Raman spectroscopy. The spectral differences between different metabolic states showed that the chemical composition varied among those metabolic states. Moreover, as can be shown by the spectral differences and principal components (PCs), different species and strains of bacteria behave differently. Furthermore, a principal component analysis (PCA) combined with support vector machines (SVM) was applied to distinguish species of bacteria within the same metabolic states. Our study provides valuable data for the comparison of bacteria between different metabolic states utilizing micro-Raman spectroscopy in combination with chemometrics models.
- ItemUsing Raman spectroscopy in infection research(Heidelberg : Spektrum, 2022) Cialla-May, Dana; Rösch, Petra; Popp, JürgenRaman spectroscopy allows to analyze bacteria and other microorganisms label and destruction free. With different Raman techniques either colonies on agar plates or small structures like single bacterial cells can be analyzed allowing for their identification as well as enabling 2D and 3D information of intracellular bacteria or biofilms. Using surface enhanced Raman spectroscopy (SERS) allows detecting and identifying viruses as well as antibiotics relevant in the treatment of infections.
- ItemTuning the corona-core ratio of polyplex micelles for selective oligonucleotide delivery to hepatocytes or hepatic immune cells(Amsterdam [u.a.] : Elsevier Science, 2023) Foo, WanLing; Cseresnyés, Zoltán; Rössel, Carsten; Teng, Yingfeng; Ramoji, Anuradha; Chi, Mingzhe; Hauswald, Walter; Huschke, Sophie; Hoeppener, Stephanie; Popp, Jürgen; Schacher, Felix H.; Sierka, Marek; Figge, Marc Thilo; Press, Adrian T.; Bauer, MichaelTargeted delivery of oligonucleotides or small molecular drugs to hepatocytes, the liver's parenchymal cells, is challenging without targeting moiety due to the highly efficient mononuclear phagocyte system (MPS) of the liver. The MPS comprises Kupffer cells and specialized sinusoidal endothelial cells, efficiently clearing nanocarriers regardless of their size and surface properties. Physiologically, this non-parenchymal shield protects hepatocytes; however, these local barriers must be overcome for drug delivery. Nanocarrier structural properties strongly influence tissue penetration, in vivo pharmacokinetics, and biodistribution profile. Here we demonstrate the in vivo biodistribution of polyplex micelles formed by polyion complexation of short interfering (si)RNA with modified poly(ethylene glycol)-block-poly(allyl glycidyl ether) (PEG-b-PAGE) diblock copolymer that carries amino moieties in the side chain. The ratio between PEG corona and siRNA complexed PAGE core of polyplex micelles was chemically varied by altering the degree of polymerization of PAGE. Applying Raman-spectroscopy and dynamic in silico modeling on the polyplex micelles, we determined the corona-core ratio (CCR) and visualized the possible micellar structure with varying CCR. The results for this model system reveal that polyplex micelles with higher CCR, i.e., better PEG coverage, exclusively accumulate and thus allow passive cell-type-specific targeting towards hepatocytes, overcoming the macrophage-rich reticuloendothelial barrier of the liver.
- ItemOptical photothermal infrared spectroscopy with simultaneously acquired Raman spectroscopy for two-dimensional microplastic identification([London] : Macmillan Publishers Limited, part of Springer Nature, 2022) Böke, Julia Sophie; Popp, Jürgen; Krafft, ChristophIn recent years, vibrational spectroscopic techniques based on Fourier transform infrared (FTIR) or Raman microspectroscopy have been suggested to fulfill the unmet need for microplastic particle detection and identification. Inter-system comparison of spectra from reference polymers enables assessing the reproducibility between instruments and advantages of emerging quantum cascade laser-based optical photothermal infrared (O-PTIR) spectroscopy. In our work, IR and Raman spectra of nine plastics, namely polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate, polycarbonate, polystyrene, silicone, polylactide acid and polymethylmethacrylate were simultaneously acquired using an O-PTIR microscope in non-contact, reflection mode. Comprehensive band assignments were presented. We determined the agreement of O-PTIR with standalone attenuated total reflection FTIR and Raman spectrometers based on the hit quality index (HQI) and introduced a two-dimensional identification (2D-HQI) approach using both Raman- and IR-HQIs. Finally, microplastic particles were prepared as test samples from known materials by wet grinding, O-PTIR data were collected and subjected to the 2D-HQI identification approach. We concluded that this framework offers improved material identification of microplastic particles in environmental, nutritious and biological matrices.
- 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
- ItemUse of polymers as wavenumber calibration standards in deep-UVRR(Amsterdam [u.a.] : Elsevier Science, 2022) Pistiki, Aikaterini; Ryabchykov, Oleg; Bocklitz, Thomas W.; Rösch, Petra; Popp, JürgenDeep-UV resonance Raman spectroscopy (UVRR) allows the classification of bacterial species with high accuracy and is a promising tool to be developed for clinical application. For this attempt, the optimization of the wavenumber calibration is required to correct the overtime changes of the Raman setup. In the present study, different polymers were investigated as potential calibration agents. The ones with many sharp bands within the spectral range 400–1900 cm−1 were selected and used for wavenumber calibration of bacterial spectra. Classification models were built using a training cross-validation dataset that was then evaluated with an independent test dataset obtained after 4 months. Without calibration, the training cross-validation dataset provided an accuracy for differentiation above 99 % that dropped to 51.2 % after test evaluation. Applying the test evaluation with PET and Teflon calibration allowed correct assignment of all spectra of Gram-positive isolates. Calibration with PS and PEI leads to misclassifications that could be overcome with majority voting. Concerning the very closely related and similar in genome and cell biochemistry Enterobacteriaceae species, all spectra of the training cross-validation dataset were correctly classified but were misclassified in test evaluation. These results show the importance of selecting the most suitable calibration agent in the classification of bacterial species and help in the optimization of the deep-UVRR technique.
- 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.