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- ItemFusion of MALDI Spectrometric Imaging and Raman Spectroscopic Data for the Analysis of Biological Samples(Lausanne : Frontiers Media, 2018) Ryabchykov, Oleg; Popp, Jürgen; Bocklitz, Thomas W.Despite of a large number of imaging techniques for the characterization of biological samples, no universal one has been reported yet. In this work, a data fusion approach was investigated for combining Raman spectroscopic data with matrix-assisted laser desorption/ionization (MALDI) mass spectrometric data. It betters the image analysis of biological samples because Raman and MALDI information can be complementary to each other. While MALDI spectrometry yields detailed information regarding the lipid content, Raman spectroscopy provides valuable information about the overall chemical composition of the sample. The combination of Raman spectroscopic and MALDI spectrometric imaging data helps distinguishing different regions within the sample with a higher precision than would be possible by using either technique. We demonstrate that a data weighting step within the data fusion is necessary to reveal additional spectral features. The selected weighting approach was evaluated by examining the proportions of variance within the data explained by the first principal components of a principal component analysis (PCA) and visualizing the PCA results for each data type and combined data. In summary, the presented data fusion approach provides a concrete guideline on how to combine Raman spectroscopic and MALDI spectrometric imaging data for biological analysis.
- ItemcellSTORM - 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, RainerHigh 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.
- ItemUsing machine-learning to optimize phase contrast in a low-cost cellphone microscope(San Francisco, CA : Public Library of Science, 2018) Diederich, Benedict; Wartmann, Rolf; Schadwinkel, Harald; Heintzmann, RainerCellphones equipped with high-quality cameras and powerful CPUs as well as GPUs are widespread. This opens new prospects to use such existing computational and imaging resources to perform medical diagnosis in developing countries at a very low cost. Many relevant samples, like biological cells or waterborn parasites, are almost fully transparent. As they do not exhibit absorption, but alter the light’s phase only, they are almost invisible in brightfield microscopy. Expensive equipment and procedures for microscopic contrasting or sample staining often are not available. Dedicated illumination approaches, tailored to the sample under investigation help to boost the contrast. This is achieved by a programmable illumination source, which also allows to measure the phase gradient using the differential phase contrast (DPC) [1, 2] or even the quantitative phase using the derived qDPC approach [3]. By applying machine-learning techniques, such as a convolutional neural network (CNN), it is possible to learn a relationship between samples to be examined and its optimal light source shapes, in order to increase e.g. phase contrast, from a given dataset to enable real-time applications. For the experimental setup, we developed a 3D-printed smartphone microscope for less than 100 $ using off-the-shelf components only such as a low-cost video projector. The fully automated system assures true Koehler illumination with an LCD as the condenser aperture and a reversed smartphone lens as the microscope objective. We show that the effect of a varied light source shape, using the pre-trained CNN, does not only improve the phase contrast, but also the impression of an improvement in optical resolution without adding any special optics, as demonstrated by measurements.
- ItemCharacterisation of a novel composite SCCmec-SCCfus element in an emerging Staphylococcus aureus strain from the Arabian Gulf region(San Francisco : Public Library of Science, 2019) Senok, Abiola; Slickers, Peter; Hotzel, Helmut; Boswihi, Samar; Braun, Sascha D.; Gawlik, Darius; Müller, Elke; Nabi, Anju; Nassar, Rania; Nitschke, Hedda; Reißig, Annett; Ruppelt-Lorz, Antje; Mafofo, Joseph; Somili, Ali M.; Udo, Edet; Ehricht, Ralf; Monecke, StefanFusidic acid is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by Staphylococcus aureus. There is an increasing number of methicillin-resistant S. aureus (MRSA) strains that harbour plasmid-borne fusB/far1 or fusC that is localised on SCC elements. In this study we examined a series of related CC30-MRSA isolates from the Arabian Gulf countries that presented with SCCmec elements and fusC, including a variant that—to the best of our knowledge—has not yet formally been described. It consisted of a class B mec complex and ccrA/B-4 genes. The fusidic acid resistance gene fusC was present, but contrary to the previously sequenced element of HDE288, it was not accompanied by tirS. This element was identified in CC30 MRSA from Kuwait, Saudi Arabia and the United Arab Emirates that usually also harbour the Panton-Valentin leukocidin (PVL) genes. It was also identified in CC8 and ST834 isolates. In addition, further CC30 MRSA strains with other SCCmec VI elements harbouring fusC were found to circulate in the Arabian Gulf region. It can be assumed that MRSA strains with SCCmec elements that include fusC have a selective advantage in both hospital and community settings warranting a review of the use of topical antibiotics and indicating the necessity of reducing over-the-counter sale of antibiotics, including fusidic acid, without prescription.Fusidic acid is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by Staphylococcus aureus. There is an increasing number of methicillin-resistant S. aureus (MRSA) strains that harbour plasmid-borne fusB/far1 or fusC that is localised on SCC elements. In this study we examined a series of related CC30-MRSA isolates from the Arabian Gulf countries that presented with SCCmec elements and fusC, including a variant that—to the best of our knowledge—has not yet formally been described. It consisted of a class B mec complex and ccrA/B-4 genes. The fusidic acid resistance gene fusC was present, but contrary to the previously sequenced element of HDE288, it was not accompanied by tirS. This element was identified in CC30 MRSA from Kuwait, Saudi Arabia and the United Arab Emirates that usually also harbour the Panton-Valentin leukocidin (PVL) genes. It was also identified in CC8 and ST834 isolates. In addition, further CC30 MRSA strains with other SCCmec VI elements harbouring fusC were found to circulate in the Arabian Gulf region. It can be assumed that MRSA strains with SCCmec elements that include fusC have a selective advantage in both hospital and community settings warranting a review of the use of topical antibiotics and indicating the necessity of reducing over-the-counter sale of antibiotics, including fusidic acid, without prescription.
- ItemRaman spectroscopy follows time-dependent changes in T lymphocytes isolated from spleen of endotoxemic mice(Rockville : American Association of Immunologists, 2019) Ramoji, Anuradha; Ryabchykov, Oleg; Galler, Kerstin; Tannert, Astrid; Markwart, Robby; Requardt, Robert Pascal; Rubio, Ignacio; Bauer, Michael; Bocklitz, Thomas W.; Popp, Jürgen; Neugebauer, UteT lymphocytes (T cells) are highly specialized members of the adaptive immune system and hold the key to the understanding the hosts’ response toward invading pathogen or pathogen-associated molecular patterns such as LPS. In this study, noninvasive Raman spectroscopy is presented as a label-free method to follow LPS-induced changes in splenic T cells during acute and postacute inflammatory phases (1, 4, 10, and 30 d) with a special focus on CD4+ and CD8+ T cells of endotoxemic C57BL/6 mice. Raman spectral analysis reveals highest chemical differences between CD4+ and CD8+ T cells originating from the control and LPS-treated mice during acute inflammation, and the differences are visible up to 10 d after the LPS insult. In the postacute phase, CD4+ and CD8+ T cells from treated and untreated mice could not be differentiated anymore, suggesting that T cells largely regained their original status. In sum, the biological information obtained from Raman spectra agrees with immunological readouts demonstrating that Raman spectroscopy is a well-suited, label-free method for following splenic T cell activation in systemic inflammation from acute to postacute phases. The method can also be applied to directly study tissue sections as is demonstrated for spleen tissue one day after LPS insult.T lymphocytes (T cells) are highly specialized members of the adaptive immune system and hold the key to the understanding the hosts’ response toward invading pathogen or pathogen-associated molecular patterns such as LPS. In this study, noninvasive Raman spectroscopy is presented as a label-free method to follow LPS-induced changes in splenic T cells during acute and postacute inflammatory phases (1, 4, 10, and 30 d) with a special focus on CD4+ and CD8+ T cells of endotoxemic C57BL/6 mice. Raman spectral analysis reveals highest chemical differences between CD4+ and CD8+ T cells originating from the control and LPS-treated mice during acute inflammation, and the differences are visible up to 10 d after the LPS insult. In the postacute phase, CD4+ and CD8+ T cells from treated and untreated mice could not be differentiated anymore, suggesting that T cells largely regained their original status. In sum, the biological information obtained from Raman spectra agrees with immunological readouts demonstrating that Raman spectroscopy is a well-suited, label-free method for following splenic T cell activation in systemic inflammation from acute to postacute phases. The method can also be applied to directly study tissue sections as is demonstrated for spleen tissue one day after LPS insult.
- ItemThermal 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, RainerConfocal 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.
- ItemHighly Sensitive Detection of the Antibiotic Ciprofloxacin by Means of Fiber Enhanced Raman Spectroscopy(Basel : MDPI, 2019) Wolf, Sebastian; Frosch, Timea; Popp, Juergen; Pletz, Mathias W.; Frosch, TorstenSepsis and septic shock exhibit a rapid course and a high fatality rate. Antibiotic treatment is time-critical and precise knowledge of the antibiotic concentration during the patients’ treatment would allow individual dose adaption. Over- and underdosing will increase the antimicrobial efficacy and reduce toxicity. We demonstrated that fiber enhanced Raman spectroscopy (FERS) can be used to detect very low concentrations of ciprofloxacin in clinically relevant doses, down to 1.5 µM. Fiber enhancement was achieved in bandgap shifted photonic crystal fibers. The high linearity between the Raman signals and the drug concentrations allows a robust calibration for drug quantification. The needed sample volume was very low (0.58 µL) and an acquisition time of 30 s allowed the rapid monitoring of ciprofloxacin levels in a less invasive way than conventional techniques. These results demonstrate that FERS has a high potential for clinical in-situ monitoring of ciprofloxacin levels.
- ItemMultimode Fabry-Perot Interferometer Probe based on Vernier Effect for Enhanced Temperature Sensing(Basel : MDPI, 2019) Gomes, André D.; Becker, Martin; Dellith, Jan; Zibaii, Mohammad Ismail; Latifi, Hamid; Rothhardt, Manfred; Bartelt, Hartmut; Frazão, OrlandoNew miniaturized sensors for biological and medical applications must be adapted to the measuring environments and they should provide a high measurement resolution to sense small changes. The Vernier effect is an effective way of magnifying the sensitivity of a device, allowing for higher resolution sensing. We applied this concept to the development of a small-size optical fiber Fabry–Perot interferometer probe that presents more than 60-fold higher sensitivity to temperature than the normal Fabry–Perot interferometer without the Vernier effect. This enables the sensor to reach higher temperature resolutions. The silica Fabry–Perot interferometer is created by focused ion beam milling of the end of a tapered multimode fiber. Multiple Fabry–Perot interferometers with shifted frequencies are generated in the cavity due to the presence of multiple modes. The reflection spectrum shows two main components in the Fast Fourier transform that give rise to the Vernier effect. The superposition of these components presents an enhancement of sensitivity to temperature. The same effect is also obtained by monitoring the reflection spectrum node without any filtering. A temperature sensitivity of −654 pm/°C was obtained between 30 °C and 120 °C, with an experimental resolution of 0.14 °C. Stability measurements are also reported.
- ItemSurface deep profile synchrotron studies of mechanically modified top-down silicon nanowires array using ultrasoft X-ray absorption near edge structure spectroscopy(Berlin : Nature Publishing, 2019) Turishchev, S.Yu.; Parinova, V.E.; Pisliaruka, Aleksandra; Koyuda, D.A.; Yermukhamed, Dana; Ming, Tingsen; Ovsyannikov, Ruslan; Smirnov, Dmitriy; Makarova, Anna; Sivakov, VladimirAtomic, electronic structure and composition of top-down metal-assisted wet-chemically etched silicon nanowires were studied by synchrotron radiation based X-ray absorption near edge structure technique. Local surrounding of the silicon and oxygen atoms in silicon nanowires array was studied on as-prepared nanostructured surfaces (atop part of nanowires) and their bulk part after, first time applied, in-situ mechanical removal atop part of the formed silicon nanowires. Silicon suboxides together with disturbed silicon dioxide were found in the composition of the formed arrays that affects the electronic structure of silicon nanowires. The results obtained by us convincingly testify to the homogeneity of the phase composition of the side walls of silicon nanowires and the electronic structure in the entire length of the nanowire. The controlled formation of the silicon nanowires array may lead to smart engineering of its atomic and electronic structure that influences the exploiting strategy of metal-assisted wet-chemically etched silicon nanowires as universal matrices for different applications.
- 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.