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DDC: 620 × Publisher: San Francisco : Public Library of Science ×

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Now showing 1 - 4 of 4
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    Raman and infrared spectroscopy reveal that proliferating and quiescent human fibroblast cells age by biochemically similar but not identical processes
    (San Francisco : Public Library of Science, 2018) Eberhardt, Katharina; Matthäus, Christian; Marthandan, Shiva; Diekmann, Stephan; Popp, Jürgen
    Dermal fibroblast cells can adopt different cell states such as proliferation, quiescence, apoptosis or senescence, in order to ensure tissue homeostasis. Proliferating (dividing) cells pass through the phases of the cell cycle, while quiescent and senescent cells exist in a non-proliferating cell cycle-arrested state. However, the reversible quiescence state is in contrast to the irreversible senescence state. Long-term quiescent cells transit into senescence indicating that cells age also when not passing through the cell cycle. Here, by label-free in vitro vibrational spectroscopy, we studied the biomolecular composition of quiescent dermal fibroblast cells and compared them with those of proliferating and senescent cells. Spectra were examined by multivariate statistical analysis using a PLS-LDA classification model, revealing differences in the biomolecular composition between the cell states mainly associated with protein alterations (variations in the side chain residues of amino acids and protein secondary structure), but also within nucleic acids and lipids. We observed spectral changes in quiescent compared to proliferating cells, which increased with quiescence cultivation time. Raman and infrared spectroscopy, which yield complementary biochemical information, clearly distinguished contact-inhibited from serum-starved quiescent cells. Furthermore, the spectra displayed spectral differences between quiescent cells and proliferating cells, which had recovered from quiescence. This became more distinct with increasing quiescence cultivation time. When comparing proliferating, (in particular long-term) quiescent and senescent cells, we found that Raman as well as infrared spectroscopy can separate these three cellular states from each other due to differences in their biomolecular composition. Our spectroscopic analysis shows that proliferating and quiescent fibroblast cells age by similar but biochemically not identical processes. Despite their aging induced changes, over long time periods quiescent cells can return into the cell cycle. Finally however, the cell cycle arrest becomes irreversible indicating senescence.Dermal fibroblast cells can adopt different cell states such as proliferation, quiescence, apoptosis or senescence, in order to ensure tissue homeostasis. Proliferating (dividing) cells pass through the phases of the cell cycle, while quiescent and senescent cells exist in a non-proliferating cell cycle-arrested state. However, the reversible quiescence state is in contrast to the irreversible senescence state. Long-term quiescent cells transit into senescence indicating that cells age also when not passing through the cell cycle. Here, by label-free in vitro vibrational spectroscopy, we studied the biomolecular composition of quiescent dermal fibroblast cells and compared them with those of proliferating and senescent cells. Spectra were examined by multivariate statistical analysis using a PLS-LDA classification model, revealing differences in the biomolecular composition between the cell states mainly associated with protein alterations (variations in the side chain residues of amino acids and protein secondary structure), but also within nucleic acids and lipids. We observed spectral changes in quiescent compared to proliferating cells, which increased with quiescence cultivation time. Raman and infrared spectroscopy, which yield complementary biochemical information, clearly distinguished contact-inhibited from serum-starved quiescent cells. Furthermore, the spectra displayed spectral differences between quiescent cells and proliferating cells, which had recovered from quiescence. This became more distinct with increasing quiescence cultivation time. When comparing proliferating, (in particular long-term) quiescent and senescent cells, we found that Raman as well as infrared spectroscopy can separate these three cellular states from each other due to differences in their biomolecular composition. Our spectroscopic analysis shows that proliferating and quiescent fibroblast cells age by similar but biochemically not identical processes. Despite their aging induced changes, over long time periods quiescent cells can return into the cell cycle. Finally however, the cell cycle arrest becomes irreversible indicating senescence.
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    Anti-correlation of HER2 and focal adhesion complexes in the plasma membrane
    (San Francisco : Public Library of Science, 2020) Weinberg, F.; Han, M.K.L.; Dahmke, I.N.; Campo, A.D.; de Jonge, N.
    Excess presence of the human epidermal growth factor receptor 2 (HER2) as well as of the focal adhesion protein complexes are associated with increased proliferation, migratory, and invasive behavior of cancer cells. A cross-regulation between HER2 and integrin signaling pathways has been found, but the exact mechanism remains elusive. Here, we investigated whether HER2 colocalizes with focal adhesion complexes on breast cancer cells overexpressing HER2. For this purpose, vinculin or talin green fluorescent protein (GFP) fusion proteins, both key constituents of focal adhesions, were expressed in breast cancer cells. HER2 was either extracellularly or intracellularly labeled with fluorescent quantum dots nanoparticles (QDs). The cell-substrate interface was analyzed at the location of the focal adhesions by means of total internal reflection fluorescent microscopy or correlative fluorescence- and scanning transmission electron microscopy. Expression of HER2 at the cell-substrate interface was only observed upon intracellular labeling, and was heterogeneous with both HER2-enriched and -low regions. In contrast to an expected enrichment of HER2 at focal adhesions, an anti-correlated expression pattern was observed for talin and HER2. Our findings suggest a spatial anti-correlation between HER2 and focal adhesion complexes for adherent cells.
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    Thermal illumination limits in 3D Raman microscopy: A comparison of different sample illumination strategies to obtain maximum imaging speed
    (San Francisco : Public Library of Science, 2019) Hauswald, Walter; Förster, Ronny; Popp, Jürgen; Heintzmann, Rainer
    Confocal Raman microscopy is a powerful tool for material science and biomedical research. However, the low Raman scattering cross-section limits the working speed, which reduces the applicability for large and sensitive samples. Here, we discuss the fundamental physical limits of Raman spectroscopy with respect to signal-to-noise, sample load and how to achieve maximal imaging speed. For this, we develop a simple model to describe arbitrary far field light microscopes and their thermal influence on the sample. This model is used to compare the practical applicability of point- and line-confocal microscopes as well as wide-field-, light sheet- and light line illumination, for the measurement of 3D biological samples. The parallelization degree of the illumination can positively affect the imaging speed as long as it is not limited by thermal sample heating. In case of heat build-up inside the sample, the advantages of parallelization can be lost due to the required attenuation of excitation and the working speed can drop below that of a sequential method. We show that for point like illumination, the exposure time is thermally not as critical for the sample as the irradiance, while for volume like illumination, the exposure time and irradiance result in the same thermal effect. The results of our theoretical study are experimentally confirmed and suggest new concepts of Raman microscopy, thus extending its applicability. The developed model can be applied to Raman imaging as well as to other modes (e.g. two- or three- photon imaging, STED, PALM/STORM, MINFLUX) where thermal effects impose a practical limit due to the high irradiance required.
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    cellSTORM - Cost-effective Super-Resolution on a Cellphone using dSTORM
    (San Francisco : Public Library of Science, 2019) Diederich, Benedict; Then, Patrick; Jügler, Alexander; Förster, Ronny; Heintzmann, Rainer
    High optical resolution in microscopy usually goes along with costly hardware components, such as lenses, mechanical setups and cameras. Several studies proved that Single Molecular Localization Microscopy can be made affordable, relying on off-the-shelf optical components and industry grade CMOS cameras. Recent technological advantages have yielded consumer-grade camera devices with surprisingly good performance. The camera sensors of smartphones have benefited of this development. Combined with computing power smartphones provide a fantastic opportunity for “imaging on a budget”. Here we show that a consumer cellphone is capable of optical super-resolution imaging by (direct) Stochastic Optical Reconstruction Microscopy (dSTORM), achieving optical resolution better than 80 nm. In addition to the use of standard reconstruction algorithms, we used a trained image-to-image generative adversarial network (GAN) to reconstruct video sequences under conditions where traditional algorithms provide sub-optimal localization performance directly on the smartphone. We believe that “cellSTORM” paves the way to make super-resolution microscopy not only affordable but available due to the ubiquity of cellphone cameras.High optical resolution in microscopy usually goes along with costly hardware components, such as lenses, mechanical setups and cameras. Several studies proved that Single Molecular Localization Microscopy can be made affordable, relying on off-the-shelf optical components and industry grade CMOS cameras. Recent technological advantages have yielded consumer-grade camera devices with surprisingly good performance. The camera sensors of smartphones have benefited of this development. Combined with computing power smartphones provide a fantastic opportunity for “imaging on a budget”. Here we show that a consumer cellphone is capable of optical super-resolution imaging by (direct) Stochastic Optical Reconstruction Microscopy (dSTORM), achieving optical resolution better than 80 nm. In addition to the use of standard reconstruction algorithms, we used a trained image-to-image generative adversarial network (GAN) to reconstruct video sequences under conditions where traditional algorithms provide sub-optimal localization performance directly on the smartphone. We believe that “cellSTORM” paves the way to make super-resolution microscopy not only affordable but available due to the ubiquity of cellphone cameras.