Quantification of Polystyrene Uptake by Different Cell Lines Using Fluorescence Microscopy and Label-Free Visualization of Intracellular Polystyrene Particles by Raman Microspectroscopic Imaging.

Environmental pollution caused by plastic is a present problem. Polystyrene is a widely used packaging material (e.g., Styrofoam) that can be broken down into microplastics through abrasion. Once the plastic is released into the environment, it is dispersed by wind and atmospheric dust. In this study, we investigated the uptake of polystyrene particles into human cells using A549 cells as a model of the alveolar epithelial barrier, CaCo-2 cells as a model of the intestinal epithelial barrier, and THP-1 cells as a model of immune cells to simulate a possible uptake of microplastics by inhalation, oral uptake, and interaction with the cellular immune system, respectively. The uptake of fluorescence-labeled beads by the different cell types was investigated by confocal laser scanning microscopy in a semi-quantitative, concentration-dependent manner. Additionally, we used Raman spectroscopy as a complementary method for label-free qualitative detection and the visualization of polystyrene within cells. The uptake of polystyrene beads by all investigated cell types was detected, while the uptake behavior of professional phagocytes (THP-1) differed from that of adherent epithelial cells.

The effects of photoactivated ciprofloxacin and bile acids on biofilms on bile duct catheters.

OBJECTIVES: This study examined the potential of a novel photoactivatable ciprofloxacin to act against bacterial infections and microbiomes related to biliary diseases. It also evaluated treatment by combining the impact of bile acids and antibiotics on biofilms. Innovative strategies were evaluated to address the elusive bile duct microbiome resulting in biofilm-related infections linked to biliary catheters. The healthy biliary system is considered sterile, but bile microbiomes can occur in disease, and these correlate with hepatobiliary diseases. Causes include biofilms that form on internal-external biliary drainage catheters. These biliary catheters were used to noninvasively study the otherwise elusive bile microbiome for a pilot study. METHODS: A new photoactivatable antibiotic was tested for efficacy against human-derived pathogenic bacterial isolates - Salmonella enterica and Escherichia coli - and catheter-derived bile duct microbiomes. In addition, the effect of bile acids on the antibiotic treatment of biofilms was quantified using crystal violet staining, confocal laser scanning microscopy, and biofilm image analysis. Two novel approaches for targeting biliary biofilms were tested. RESULTS: A photoactivated antibiotic based on ciprofloxacin showed efficacy in preventing biofilm formation and reducing bacterial viability without harming eukaryotic cells. Furthermore, combination treatment of antibiotics with bile acids, such as ursodesoxycholic acid, mildly influenced biofilm biomass but reduced bacterial survival within biofilms. CONCLUSION: Bile acids, in addition to their endocrine and paracrine functions, may enhance antibiotic killing of bacterial biofilms compared with antibiotics alone. These approaches hold promise for treating biliary infections such as cholangitis.

Findaureus: An open-source application for locating Staphylococcus aureus in fluorescence-labelled infected bone tissue slices.

Staphylococcus aureus (S. aureus) is a facultative pathogenic bacterium that can cause infections in various tissue types in humans. Fluorescence imaging techniques have been employed to visualize the bacteria in ex-vivo samples mostly in research, aiding in the understanding of the etiology of the pathogen. However, the multispectral images generated from fluorescence microscopes are complex, making it difficult to locate bacteria across image files, especially in consecutive planes with different imaging depths. To address this issue, we present Findaureus, an open-source application specifically designed to locate and extract critical information about bacteria, especially S. aureus. Due to the limited availability of datasets, we tested the application on a dataset comprising fluorescence-labelled infected mouse bone tissue images, achieving an accuracy of 95%. We compared Findaureus with other state-of-the-art image analysis tools and found that it performed better, given its specificity toward bacteria localization. The proposed approach has the potential to aid in medical research of bone infections and can be extended to other tissue and bacteria types in the future.

Insights into S. aureus-Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging.

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation.

Lysosome-targeting pH indicator based on peri-fused naphthalene monoimide with superior stability for long term live cell imaging.

Lysosomes, the acidic degradation compartments of eukaryotic cells, play an essential role in many physiological processes. Their dysfunction is associated with a number of diseases, which are often related to an altered localization or luminal pH. Thus, the in-depth characterization of lysosomes within the intact eukaryotic cell is of utmost interest. For microscopic evaluation of lysosomal distribution and acidity, a number of labels have been developed, but many showed poor organelle specificity or rapid clearing from lysosomes, rendering them unsuitable for long-term observations. Here, we describe the synthesis and spectroscopic properties of a novel small molecule marker for lysosomes based on naphthalene monoimide with reversible, pH-dependent spectral shifts in both the absorption and the emission spectrum and acidity-associated changes in fluorescence lifetime. The dye can be excited either with single- or two-photon excitation and appears to be very stably associated with lysosomes for several days. We used this chromophore to detect chemically-induced changes of lysosomal pH in HeLa cells by ratiometric and FLIM imaging.

Towards Bacteria Counting in DI Water of Several Microliters or Growing Suspension Using Impedance Biochips.

We counted bacterial cells of E. coli strain K12 in several-microliter DI water or in several-microliter PBS in the low optical density (OD) range (OD = 0.05-1.08) in contact with the surface of Si-based impedance biochips with ring electrodes by impedance measurements. The multiparameter fit of the impedance data allowed calibration of the impedance data with the concentration cb of the E. coli cells in the range of cb = 0.06 to 1.26 × 109 cells/mL. The results showed that for E. coli in DI water and in PBS, the modelled impedance parameters depend linearly on the concentration of cells in the range of cb = 0.06 to 1.26 × 109 cells/mL, whereas the OD, which was independently measured with a spectrophotometer, was only linearly dependent on the concentration of the E. coli cells in the range of cb = 0.06 to 0.50 × 109 cells/mL.

A new human adipocyte model with PTEN haploinsufficiency.

Few human cell strains are suitable and readily available as in vitro adipocyte models. We used resected lipoma tissue from a patient with germline phosphatase and tensin homolog (PTEN) haploinsufficiency to establish a preadipocyte cell strain termed LipPD1 and aimed to characterize cellular functions and signalling pathway alterations in comparison to the established adipocyte model Simpson-Golabi-Behmel-Syndrome (SGBS) and to primary stromal-vascular fraction cells. We found that both cellular life span and the capacity for adipocyte differentiation as well as adipocyte-specific functions were preserved in LipPD1 and comparable to SGBS adipocytes. Basal and growth factor-stimulated activation of the PI3 K/AKT signalling pathway was increased in LipPD1 preadipocytes, corresponding to reduced PTEN levels in comparison to SGBS cells. Altogether, LipPD1 cells are a novel primary cell model with a defined genetic lesion suitable for the study of adipocyte biology.

Raman Spectroscopy Follows Time-Dependent Changes in T Lymphocytes Isolated from Spleen of Endotoxemic Mice.

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.

Phenotypic antibiotic susceptibility testing of pathogenic bacteria using photonic readout methods: recent achievements and impact.

The development of antibiotic resistances in common pathogens is an increasing challenge for therapy of infections and especially severe complications like sepsis. To prevent administration of broad-spectrum and potentially non-effective antibiotics, the susceptibility spectrum of the pathogens underlying the infection has to be determined. Current phenotypic standard methods for antibiotic susceptibility testing (AST) require the isolation of pathogens from the patient and the subsequent culturing in the presence of antibiotics leading to results only after 24-72 h. Since the early initialization of an effective antibiotic therapy is crucial for positive treatment result in severe infections, faster methods of AST are urgently needed. A large number of different assay systems are currently tested for their practicability for fast detection of antibiotic resistance profiles. They can be divided into genotypic ones which detect the presence of certain genes or gene products associated with resistances and phenotypic assays which determine the effect of antibiotics on the pathogens. In this mini-review, we summarize current developments in fast phenotypic tests that use photonic approaches and critically discuss their status. We further outline steps that are required to bring these assays into clinical practice.

Microfluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. coli under Antibiotic Stress.

Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments.

Photonic monitoring of treatment during infection and sepsis: development of new detection strategies and potential clinical applications.

Despite the strong decline in the infection-associated mortality since the development of the first antibiotics, infectious diseases are still a major cause of death in the world. With the rising number of antibiotic-resistant pathogens, the incidence of deaths caused by infections may increase strongly in the future. Survival rates in sepsis, which occurs when body response to infections becomes uncontrolled, are still very poor if an adequate therapy is not initiated immediately. Therefore, approaches to monitor the treatment efficacy are crucially needed to adapt therapeutic strategies according to the patient's response. An increasing number of photonic technologies are being considered for diagnostic purpose and monitoring of therapeutic response; however many of these strategies have not been introduced into clinical routine, yet. Here, we review photonic strategies to monitor response to treatment in patients with infectious disease, sepsis, and septic shock. We also include some selected approaches for the development of new drugs in animal models as well as new monitoring strategies which might be applicable to evaluate treatment response in humans in the future. Figure Label-free probing of blood properties using photonics.

Lipid dynamics in boar sperm studied by advanced fluorescence imaging techniques.

The (re)organization of membrane components is of special importance to prepare mammalian sperm to fertilization. Establishing suitable methods to examine physico-chemical membrane parameters is of high interest. We characterized the behavior of fluorescent (NBD) analogs of sphingomyelin (SM), phosphatidylserine (PS), and cholesterol (Ch) in the acrosomal and postacrosomal macrodomain of boar sperm. Due to their specific transverse membrane distribution, a leaflet-specific investigation of membrane properties is possible. The behavior of lipid analogs in boar sperm was investigated by fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS). The results were compared with regard to the different temporal and spatial resolution of the methods. For the first time, fluorescence lifetimes of lipid analogs were determined in sperm cell membrane and found to be in a range characteristic for the liquid-disordered phase in artificial lipid membranes. FLIM analyses further indicate a more fluid microenvironment of NBD-Ch and NBD-PS in the postacrosomal compared to the acrosomal region. The concept of a more fluid cytoplasmic leaflet is supported by lower fluorescence lifetime and higher average D values (FCS) for NBD-PS in both head compartments. Whereas FLIM analyses did not indicate coexisting distinct liquid-ordered and -disordered domains in any of the head regions, comparisons between FRAP and FCS measurements suggest the incorporation of NBD-SM as well as NBD-PS in postacrosomal subpopulations with different diffusion velocity. The analog-specific results indicate that the lipid analogs used are suitable to report on the various physicochemical properties of different microenvironments.

Cholesterol sensitises the transient receptor potential channel TRPV3 to lower temperatures and activator concentrations.

TRPV3, a thermosensitive cation channel, is predominantly expressed in keratinocytes. It contributes to physiological processes such as thermosensation, nociception, and skin development. TRPV3 is polymodally regulated by chemical agonists, innocuous heat, intracellular acidification or by membrane depolarization. By manipulating the content of plasma membrane cholesterol, a key modulator of the physicochemical properties of biological membranes, we here addressed the question, how the lipid environment influences TRPV3. Cholesterol supplementation robustly potentiated TRPV3 channel activity by sensitising it to lower concentrations of chemical activators. In addition, the thermal activation of TRPV3 is significantly shifted to lower temperatures in cholesterol-enriched cells. The sensitising effect of cholesterol was not caused by an increased plasma membrane targeting of the channel. In HaCaT keratinocytes, which natively express TRPV3, a cholesterol-mediated sensitisation of TRPV3-like responses was reproduced. The cholesterol-dependent modulation of TRPV3 activity may provide a molecular mechanism to interpret its involvement in keratinocyte differentiation.

HaCaT keratinocytes exhibit a cholesterol and plasma membrane viscosity gradient during directed migration.

Keratinocyte migration plays an important role in cutaneous wound healing by supporting the process of reepithelialisation. During directional migration cells develop a polarised shape with an asymmetric distribution of a variety of signalling molecules in their plasma membrane. Here, we investigated front-to-back differences of the physical properties of the plasma membrane of migrating keratinocyte-like HaCaT cells. Using FRAP and fluorescence lifetime analysis, both under TIR illumination, we demonstrate a reduced viscosity of the plasma membrane in the lamellipodia of migrating HaCaT cells compared with the cell rears. This asymmetry is most likely caused by a reduced cholesterol content of the lamellipodia as demonstrated by filipin staining. siRNA-mediated silencing of the cholesterol transporter ABCA1, which is known to redistribute cholesterol from rafts to non-raft regions, as well as pharmacological inhibition of this transporter with glibenclamide, strongly diminished the viscosity gradient of the plasma membrane. In addition, HaCaT cell migration was inhibited by glibenclamide treatment. These data suggest a preferential role of non-raft cholesterol in the establishment of the asymmetric plasma membrane viscosity.

Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells.

In recent years, human dendritic cells (DCs) could be subdivided into CD304+ plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter encompassing the CD1c+, CD16+, and CD141+ DC subsets. To date, the low frequency of these DCs in human blood has essentially prevented functional studies defining their specific contribution to antigen presentation. We have established a protocol for an effective isolation of pDC and cDC subsets to high purity. Using this approach, we show that CD141+ DCs are the only cells in human blood that express the chemokine receptor XCR1 and respond to the specific ligand XCL1 by Ca2+ mobilization and potent chemotaxis. More importantly, we demonstrate that CD141+ DCs excel in cross-presentation of soluble or cell-associated antigen to CD8+ T cells when directly compared with CD1c+ DCs, CD16+ DCs, and pDCs from the same donors. Both in their functional XCR1 expression and their effective processing and presentation of exogenous antigen in the context of major histocompatibility complex class I, human CD141+ DCs correspond to mouse CD8+ DCs, a subset known for superior antigen cross-presentation in vivo. These data define CD141+ DCs as professional antigen cross-presenting DCs in the human.

Localisation of endothelin B receptor variants to plasma membrane microdomains and its effects on downstream signalling.

The endothelin B (ET(B)) receptor can undergo a proteolytic cleavage resulting in an unglycosylated N-terminally truncated receptor. We investigated whether ET(B) receptor processing affects caveolar localisation and mitogenic signalling. Distinct subcellular localisations of ET(B) receptor constructs and epidermal growth factor (EGF) receptor ligands were analysed performing detergent-free caveolae preparations and total internal reflection fluorescence microscopy. ET(B) receptor-induced transactivation of the EGF receptor and its downstream signalling was investigated performing shedding assays and ERK1/2 phosphorylation analyses. In COS7 cells, the N-terminally truncated but not the full-length or glycosylation-deficient ET(B) receptor localised to caveolae. In caveolae-free HEK293 cells, only ET(B) receptor constructs fused to caveolin-2 localised to membrane microdomains. A caveolar accumulation of the ET(B) receptor disfavoured EGF receptor ligand shedding. Nonetheless, the activation of ERK1/2 was efficient and long-lasting. In HEK293 cells, the shedding activity was also impaired by N-terminal truncation. The subsequent ERK1/2 phosphorylation was long-lasting only for the full-length ET(B) receptor. We conclude that the ET(B) receptor localisation might depend on the presence of caveolae within the cell investigated. The data further suggest that caveolar enrichment of ET(B) receptors does not facilitate the release of EGF receptor ligands. However, independent of their localisation, ET(B) receptors are able to induce an ERK1/2 phosphorylation.

Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes.

Proline-rich sequences (PRS) and their recognition domains have emerged as transposable protein interaction modules during eukaryotic evolution. They are especially abundant in proteins associated with pre-mRNA splicing and likely assist in the formation of the spliceosome by binding to GYF and WW domains. Here we profile PRS-mediated interactions of the CD2BP2/52K GYF domain by a site-specific peptide inhibitor and stable isotope labeling/mass spectrometry analysis. Several PRS hubs with multiple proline-rich motifs exist that can recruit GYF and/or WW domains. Saturating the PRS sites by an isolated GYF domain inhibited splicing at the level of A complex formation. The interactions mediated by PRS are therefore important to the early phases of spliceosomal assembly.

Convolution-based one and two component FRAP analysis: theory and application.

The method of fluorescence redistribution after photobleaching (FRAP) is increasingly receiving interest in biological applications as it is nowadays used not only to determine mobility parameters per se, but to investigate dynamic changes in the concentration or distribution of diffusing molecules. Here, we develop a new simple convolution-based approach to analyze FRAP data using the whole image information. This method does not require information about the timing and localization of the bleaching event but uses the first image acquired directly after photobleaching to calculate the intensity distributions, instead. Changes in pools of molecules with different velocities, which are monitored by applying repetitive FRAP experiments within a single cell, can be analyzed by means of a global model by assuming two global diffusion coefficients with changing portions. We validate the approach by simulation and show that translocation of the YFP-fused PH-domain of phospholipase Cdelta1 can be quantitatively monitored by FRAP analysis in a time-resolved manner. The new FRAP data analysis procedure may be applied to investigate signal transduction pathways using biosensors that change their mobility. An altered mobility in response to the activation of signaling cascades may result either from an altered size of the biosensor, e.g. due to multimerization processes or from translocation of the sensor to an environment with different viscosity.

Signal amplification between Gbetagamma release and PI3Kgamma-mediated PI(3,4,5)P3 formation monitored by a fluorescent Gbetagamma biosensor protein and repetitive two component total internal reflection/fluorescence redistribution after photobleaching analysis.

Phosphoinositide 3-kinase gamma (PI3Kgamma) is activated by Gbetagamma release after stimulation of Galpha i -coupled receptors, involving a recruitment of the enzyme to the plasma membrane via interaction of the regulatory subunit p101 or p87 with Gbetagamma. The receptor-mediated release of Gbetagamma was, however, insufficient to elicit a translocation of p101 observable by classical fluorescence microscopy approaches. Since the mobilities of plasma membrane-associated and cytosolic proteins differ strongly, small changes in the amount of plasma membrane association should be detectable by an altered diffusional behavior. Here, changes in mobility were monitored by fluorescence redistribution after photobleaching (FRAP) which was repetitively applied before and after stimulation of cells. To combine the advantages of total internal reflection (TIR) illumination, which preferentially excites fluorophors located at or near the plasma membrane, with that provided by the mobility information, we developed a combined TIR/FRAP setup which enabled us to point bleach parts of an image that was observed under TIR illumination. For FRAP data analysis, we introduce a convolution-based method and a global two component model. Using this TIR/FRAP approach, an increased plasma membrane association of the fluorescent Gbetagamma-binding domain of p101 after Gbetagamma release by G protein-coupled receptor stimulation could be detected and quantified. By comparing the translocation efficiency of this domain with that of YFP-GRP1(PH), a biosensor for the PI3Kgamma product PI(3,4,5)P3, we evaluate the signal amplification between Gbetagamma release and PI(3,4,5)P3 formation after activation of Galpha i -coupled receptors.

The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability.

The bovine seminal plasma protein PDC-109 exerts an essential influence on the sperm cell plasma membrane during capacitation. However, by any mechanism, it has to be ensured that this function of the protein on sperm cells is not initiated too early, that is, upon ejaculation when PDC-109 and sperm cells come into first contact, but rather at later stages of sperm genesis in the female genital tract. To answer the question of whether changes of the bovine sperm lipid composition can modulate the effect of PDC-109 on sperm membranes, we have investigated the influence of PDC-109 on the integrity of (i) differently composed lipid vesicles and of (ii) membranes from human red blood cells and bovine spermatozoa. PDC-109 most effectively disturbed lipid membranes composed of choline-containing phospholipids and in the absence of cholesterol. The impact of the protein on lipid vesicles was attenuated in the presence of cholesterol or of noncholine-containing phospholipids, such as phosphatidylethanolamine or phosphatidylserine. An extraction of cholesterol from lipid or biological membranes using methyl-beta-cyclodextrin caused an increased membrane perturbation by PDC-109. Our results argue for a oppositional effect of PDC-109 during sperm cell genesis. We hypothesize that the lipid composition of ejaculated bull sperm cells allows a binding of PDC-109 without leading to an impairment of the plasma membrane. At later stages of sperm cell genesis upon release of cholesterol from sperm membranes, PDC-109 triggers a destabilization of the cells.