Klinisch-spektroskopische Diagnostik

Arbeitsgruppe Prof. Dr. Ute Neugebauer

Ute Neugebauer, Univ.-Prof. Dr.

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Mitarbeiter der AG Neugebauer

  1. Neugebauer, Ute, Univ.-Prof. Dr.

    Professur Klinisch-Spektroskopische Diagnostik
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47 Publikationen filtern

Die Publikationen filtern

  1. Unraveling N-FINDR: A generalization of geometrical linear unmixing approaches under the pure pixel assumption

    Autoren
    R. Guliev, U. Neugebauer, C. Beleites
    Erscheinungsjahr
    Erschienen in:
    Analytica chimica acta: an international journal devoted to all branches of analytical chemistry
    Background. Hyperspectral unmixing is an important task in hyperspectral image analysis, aiming to decompose mixed pixels into pure spectral signatures. Among existing unmixing approaches, the N-FINDR algorithm is one of the most widely used methods. However, over time, multiple implementations have emerged in the literature, often differing in parameter choices, algorithmic details, and operational assumptions. This lack of standardization has led to confusion and difficulties in comparing studies. As a result, the field lacks a unified and unambiguous formulation of the N-FINDR algorithm that clearly defines its principles, its relationship to closely related methods, and enables reproducible research. Results. We introduce a generalized and standardized formulation of the N-FINDR algorithm that explicitly defines its geometric principles, parameter choices, and operational steps. Ambiguous abbreviations commonly used in prior descriptions are replaced with explicit parameter values, enabling transparent interpretation and consistent implementation. The proposed framework clarifies the relationship between N-FINDR and other pure-pixel endmember extraction methods, particularly Vertex Component Analysis (VCA), highlighting shared assumptions and key differences. Comprehensive benchmarking experiments evaluate the influence of algorithmic variants, initialization strategies, and parameter settings on extraction performance. Based on this analysis, we identify best-practice configurations that provide robust and reproducible results across datasets. To facilitate adoption, we release open-source Python and R packages implementing the recommended variants, allowing both expert and non-expert users to apply N-FINDR reliably without detailed algorithmic tuning. Significance and Novelty. This work resolves long-standing ambiguities surrounding the N-FINDR algorithm by providing a standardized, transparent, and reproducible formulation. By clarifying its relationship to other pure-pixel methods and establishing best practices through systematic benchmarking, the study improves the reliability of hyperspectral unmixing research. The availability of open-source implementations further lowers the barrier to entry, enabling broader and more consistent use of N-FINDR across the hyperspectral imaging community.
    Universitätsbibliographie Jena:
    fsu_mods_00036441Externer Link

  2. Conceptual Differences Between Hyperspectral Unmixing, PCA, and Clustering: A Practical Demonstration Using Single-Cell Raman Imaging

    Autoren
    R. Guliev, U. Neugebauer
    Erscheinungsjahr
    Erschienen in:
    Journal of chemometrics : a journal of the Chemometrics Society
    Hyperspectral unmixing is a well-established approach for analyzing spectroscopic images by recovering chemically interpretable component spectra and their spatial distributions. Despite its distinct analytical objective, unmixing is frequently discussed alongside or directly compared with more familiar multivariate techniques such as principal component analysis (PCA) and clustering, which address fundamentally different questions. Although these conceptual distinctions have long been recognized in chemometrics and hyperspectral imaging, they are often treated implicitly and are not always demonstrated side by side in a single, accessible framework, particularly for nonexpert users of spectroscopic imaging methods. In this work, we provide a concise qualitative illustration of these established principles using a Raman spectroscopic image of a single eukaryotic cell. Employing classical implementations of PCA, k-means and fuzzy c-means clustering, and hyperspectral unmixing based on the N-FINDR end-member extraction algorithm, we show how unmixing yields chemically interpretable pure component spectra and abundance maps, whereas PCA and clustering produce complementary but fundamentally different types of outputs. This contribution serves as a practical reference, clarifying the principles of unmixing and supporting its proper application and communication in spectroscopic studies.
    Universitätsbibliographie Jena:
    fsu_mods_00036753Externer Link

  3. Insights into chlamydial infection at the sub-cellular level using label-free Raman spectroscopy in comparison to electron microscopy

    Autoren
    N. Unger, E. Liebler-Tenorio, R. Guliev, S. Eiserloh, S. Nietzsche, F. Nowak, S. Zuchantke, C. Berens, C. Schnee, U. Neugebauer
    Erscheinungsjahr
    Erschienen in:
    Journal of Biological Chemistry
    Intracellular infections are difficult to study as the host cell protects the pathogen from direct observation from the outside. Transmission electron microscopy (TEM) is the most commonly used method for subcellular analysis. However, sample preparation is based on fixation which prevents continuous observation. Here, we focus on the obligate intracellular bacterium Chlamydia abortus. It causes infections primarily in small ruminant livestock, and can also be transmitted to humans, where it can cause disease. Diagnosis is difficult, requiring PCR or cell culture. At the moment, non-invasive methods for the direct study of intracellular infections are rare and not yet established in routine analysis. In this study, we present 3D confocal Raman imaging as a non-invasive tool to investigate and characterize the infection directly inside intact host cells without the need for any purification step and compare the results to established conventional TEM. A 2D cell culture infection model with Buffalo Green Monkey kidney cells was employed and infection with Chlamydia abortus S26/3 was characterized at different time points post infection. Using multivariate statistical data analysis, high-quality false color image stacks were generated from the Raman data. Two Chlamydia morphoforms, elementary body and reticulate body, could be distinguished based on their Raman spectral features: reticulate bodies are characterized by prominent lipid and nucleic acid signals while elementary bodies revealed higher carbohydrate and protein signals. This provides complementary information to TEM analysis where morphoforms are differentiated based on size and contrast. The complementary nature of both imaging methods is discussed in the manuscript.
    Universitätsbibliographie Jena:
    fsu_mods_00029339Externer Link

  4. A comparative study of robustness to noise and interpretability in U-Net-based denoising of Raman spectra

    Autoren
    A. Mokari, S. Eiserloh, O. Ryabchykov, U. Neugebauer, T. Bocklitz
    Erscheinungsjahr
    Erschienen in:
    Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
    Universitätsbibliographie Jena:
    fsu_mods_00026011Externer Link

  5. Molecular Insights into the Heme-Binding Potential of Plant NCR247-Derived Peptides

    Autoren
    S. Vaidya, D. Rathod, A. Ramoji, U. Neugebauer, D. Imhof
    Erscheinungsjahr
    Erschienen in:
    ChemBioChem : a European journal of chemical biology
    Heme is involved in many critical processes in pathogenic bacteria as iron acquisition by these microorganisms is achieved by either direct uptake of heme or use of heme-binding proteins called hemophores. Exploring the underlying mechanisms on a molecular level can open new avenues in understanding the host-pathogen interactions. Any imbalance of the heme concentration has a direct impact on the bacterial growth and survival. Thus, heme-regulated proteins that are involved in heme homeostasis poise to be promising targets for research. Similarly, naturally occurring compounds, including cysteine-rich peptides from either plant secondary metabolites or venom toxins from vertebrates and invertebrates, have been studied for their therapeutic potential. NCR247 is such a cysteine-rich peptide, known to be crucial for nitrogenase activity in M. truncatula and its symbiotic relation with S. meliloti. NCR247-derived peptides were suggested to serve as high-affinity heme-binding molecules with remarkable heme-capturing properties. A comprehensive biochemical and computational analysis of NCR247-derived peptides, however, redefines their heme-binding capacity and consequently their potential therapeutic role.
    Universitätsbibliographie Jena:
    fsu_mods_00019376Externer Link

  7. Super-resolution fluorescence MINFLUX microscopy to study lipid dynamics in live-cell plasma membranes

    Autor
    A. Koerfer
    Erscheinungsjahr
    The Plasma Membrane (PM) of eukaryotic cells separates the intracellular environment from the extracellular space and plays a crucial role in regulating material exchange, signal transduction, and host-pathogen interactions. It consists of a heterogeneous lipid bilayer with embedded proteins, enabling localised processes such as endocytosis, exocytosis, and viral assembly. However, its small size and rapid dynamics make direct study challenging. A prominent example is the assembly and release of Human Immunodeficiency Virus type 1 (HIV-1). During viral assembly, the viral protein Gag binds the cytosolic leaflet of the PM, triggering clustering and membrane curvature that leads to budding of new virions (~120 nm). This is accompanied by local enrichment of cholesterol and saturated lipids, though the underlying mechanisms remain unclear. This thesis investigates lipid dynamics in the live-cell PM using the super-resolution technique Minimal Photon Flux (MINFLUX) microscopy. MINFLUX enables tracking of individual fluorescent lipid analogues with high spatial (nanometre) and temporal resolution. Both 2D and 3D MINFLUX tracking are used to study lipid diffusion and enrichment during the formation of HIV Virus-Like Particles (VLPs) induced by Gag. 3D MINFLUX reveals membrane topography at ~20 nm resolution, allowing classification of intermediate budding stages. Fully formed VLPs measure 130 ± 5 nm, consistent with literature. Correlating structural features with lipid enrichment and diffusion provides insights not accessible with 2D data alone. Additionally, a custom analysis workflow integrating confocal imaging MINFLUX tracking is presented, enabling quantitative correlation between lipid dynamics and Gag localisation. Overall, this work establishes an optimised MINFLUX-based workflow for studying lipid dynamics in live-cell PMs and demonstrates the importance of integrating structural information to understand membrane remodelling during viral budding.
    Universitätsbibliographie Jena:
    fsu_mods_00029829Externer Link

  8. Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis

    Autoren
    S. Mandal, R. Motganhalli Ravikumar, A. Tannert, A. Urbanek, R. Guliev, M. Naumann, S. Coldewey, U. Dahmen, L. Carvalho, L. Bastião Silva, U. Neugebauer
    Erscheinungsjahr
    Erschienen in:
    Scientific reports
    Bone tissue, with its complex structure, often necessitates decalcification of the hard tissue for ex vivo morphological studies. The choice of a suitable decalcification method plays a crucial role in preserving desired features and ensuring compatibility with diverse imaging techniques. The search for a universal decalcification method that is suitable for a range of biophotonic analyses remains an ongoing challenge. In this study, we systematically assessed five standard bone decalcification protocols, encompassing strong mineralic acids (3% and 5% nitric acid), a commercially available formulation of hydrochloric and formic acid), as well as weak organic acids (5% trichloroacetic acid and 8% formic acid), and a chelating agent (25% ethylenediamine-tetraacetic acid) with varying decalcification durations, using mouse long bones as our experimental model. Our imaging analysis panel included classical histological staining (Hematoxylin and Eosin, H&E), immunofluorescence staining, and label-free Raman microspectroscopic imaging. We used cryosections instead of paraffin sections since paraffin interferes with tissue Raman signals. This approach is not as commonly used as it is more prone to handling artifacts, but is the preferred method for subsequent Raman analysis. Decalcification efficacy was evaluated based on various qualitative and some quantitative imaging parameters by 2–3 independent observers. Our systematic approach revealed that the chelating agent, when used for 24 h, optimally preserved bone features and, thus, would be the ideal decalcifying agent for comprehensive subsequent analysis. However, the choice of decalcifier and the ideal decalcification duration may vary depending on the type and thickness of bone, necessitating tailored adjustments to meet specific experimental requirements.
    Universitätsbibliographie Jena:
    fsu_mods_00019104Externer Link

  9. Investigation of Collagen Crosslinks Introduced By a Femtosecond Laser

    Autoren
    D. Fischer, A. Stöcker, A. Tannert, T. Koch, U. Neugebauer, R. Ackermann, J. Missbach-Guentner, S. Nolte, C. Rußmann
    Erscheinungsjahr
    Erschienen in:
    2025 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC)
    Universitätsbibliographie Jena:
    fsu_mods_00027637Externer Link

  10. Characterizing Metabolic Shifts in Septic Murine Kidney Tissue Using 2P-FLIM for Early Sepsis Detection

    Autoren
    S. Greiner, M. Ebrahimi, M. Rodewald, A. Urbanek, T. Meyer-Zedler, M. Schmitt, U. Neugebauer, J. Popp
    Erscheinungsjahr
    Erschienen in:
    Bioengineering
    In this study, thin mouse kidney sections from healthy mice and those infected leading to acute and chronic sepsis were examined with two-photon excited fluorescence lifetime imaging (2P-FLIM) using the endogenous fluorescent coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). The results presented show that this approach is a powerful tool for investigating cell metabolism in thin tissue sections. An adapted measurement routine was established for these samples by performing a spectral scan, identifying a combination of two excitation wavelengths and two detection ranges suitable for detailed scan images of NADH and FAD. Selected positions in thin slices of the renal cortex of nine mice (three healthy, three with chronic sepsis, and three with acute sepsis) were studied using 2P-FLIM. In addition, overview images were obtained using two-photon excited fluorescence (2PEF) intensity. This study shows that healthy kidney slices differ considerably from those with acute sepsis with regard to their fluorescence lifetime signatures. The latter shows a difference in metabolism between the inner and outer cortex, indicating that outer cortical tubular cells switch their metabolism from oxidative phosphorylation to glycolysis in kidneys from mice with acute sepsis and back in later stages, as seen for mice with chronic infections. These findings suggest that 2P-FLIM could serve as a powerful tool for early-stage sepsis diagnosis and monitoring metabolic recovery during treatment.
    Universitätsbibliographie Jena:
    fsu_mods_00021975Externer Link

  11. Investigating alveolar macrophages in an human ex vivo precision‐cut lung slice model of SARS‐CoV‐2 infection using Raman spectroscopy: A case study

    Autoren
    M. Naumann, F. Hornung, S. Eiserloh, A. Tannert, A. Häder, R. Guliev, T. Sandhaus, S. Deinhardt‐Emmer, U. Neugebauer
    Erscheinungsjahr
    Erschienen in:
    Clinical and Translational Medicine
    Background: Alveolar macrophages (AMs) are crucial innate immune cells that play important roles during infection with severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2). Ex vivo human precision‐cut lung slices (PCLSs) are well‐suited models to study immune reactions and biochemical changes within host cells as well as to follow functional macrophage phenotype plasticity within complex tissue environment. Raman spectroscopy emerged in recent years as a powerful method for label‐free cell characterization. Methods: Human PCLSs from one donor were infected with either the SARS‐CoV‐2 delta or omicron variant. Immunofluorescence microscopy localized AMs and virus particles. Cytokine levels of interferon‐gamma (IFN‐γ) and interleukin 18 (IL‐18) were quantified. The lung slice model was optimized for label‐free Raman spectroscopic imaging and for the characterization of single AMs within the three‐dimensional structure of the PCLS model. Results: Fluorescence microscopy confirmed the location of AMs and virus particles within the PCLS model. Raman spectroscopic imaging generated false‐colour images, revealing distinct spectroscopic differences between AMs in the uninfected control PCLS model and those in PCLS models infected with SARS‐CoV‐2. These differences included variations in intracellular RNA, carotenoid, triacyl glyceride, and glucose levels, consistent in interpretation with cytokine quantification data. A linear discriminant analysis (LDA) classification model achieved an 83% accuracy in distinguishing cells from infected lung slices from those of the uninfected controls. The LDA loadings pointed to spectral bands that had been previously identified in an in vitro stimulation study of macrophages. Conclusions: Raman spectroscopy can characterize the cellular immune response and phenotype plasticity of AMs to infection with SARS‐CoV‐2 within a PCLS model in a label‐free and non‐invasive manner. The ability to distinguish cells from infected PCLSs from cells of the uninfected control PCLS based on intracellular biochemical changes highlights the potential of Raman spectroscopy as a powerful diagnostic tool in immunology and clinical diagnostics.
    Universitätsbibliographie Jena:
    fsu_mods_00030389Externer Link

  12. Long-term device stability for Raman spectroscopy

    Autoren
    S. Guo, A. Ramoji, A. Pistiki, H. Yilmaz, U. Glaser, D. Vasquez-Pinzon, I. Schie, U. Neugebauer, A. Silge, J. Popp, T. Bocklitz
    Erscheinungsjahr
    Erschienen in:
    The analyst: the analytical journal of the Royal Society of Chemistry
    Universitätsbibliographie Jena:
    fsu_mods_00025267Externer Link
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