Mathematical Modeling for 2D Light-Sheet Fluorescence Microscopy image reconstruction

We study an inverse problem for Light Sheet Fluorescence Microscopy (LSFM), where the density of fluorescent molecules needs to be reconstructed. Our first step is to present a mathematical model to describe the measurements obtained by an optic camera during an LSFM experiment. Two meaningful stages are considered: excitation and fluorescence. We propose a paraxial model to describe the excitation process which is directly related with the Fermi pencil-beam equation. For the fluorescence stage, we use the transport equation to describe the transport of photons towards the detection camera. For the mathematical inverse problem that we obtain after the modeling, we present a uniqueness result, recasting the problem as the recovery of the initial condition for the heat equation in $\mathbb{R}\times(0,\infty)$ from measurements in a space-time curve. Additionally, we present numerical experiments to recover the density of the fluorescent molecules by discretizing the proposed model and facing this problem as the solution of a large and sparse linear system. Some iterative and regularized methods are used to achieve this objective. The results show that solving the inverse problem achieves better reconstructions than the direct acquisition method that is currently used

Accuracy of single beam timing lights for determining velocities in a flying 20-m sprint: Does timing light height matter?

Background: The purpose of this study was to evaluate the accuracy of timing lights (TL) at different heights for measuring velocities during sprinting. Methods: Two sets of single beam TL were used to determine velocities reached in a flying 20-m sprint in 15 healthy and physically active male participants. In TL64, all TL were set up at a height of 64 cm, and in TL100, all TL were set up at 100 cm, respectively. Participants performed three valid trials. The recordings of high-speed video cameras were used as a reference. Results: ICC and Pearson’s r values between both timing light heights and the reference system were almost perfect (0.969–0.991). Bland & Altman’s LOA (95 %) indicated low systematic and unsystematic errors, with somewhat smaller LOA for TL100 (-0.013–0.121 m/s) than for TL64 (-0.060–0.120 m/s). Measures of between-trial reliability of running velocities showed a high relative (ICC) and absolute (RMSE) reliability, with the reference system showing slightly better values in all reliability measures (ICC=0.935; RMSE<0.001 m/s) compared to TL64 and TL100 (ICC=0.894, 0.887; RMSE=0.107 m/s, 0.124 m/s, respectively). The usefulness, determined by comparing the typical error (TE) with the smallest worthwhile change (SWC), was considered as “OK” (TE ≈ SWC) for all three systems. Conclusions: Results suggest that TL at both heights (TL64 and TL100) can be considered as accurate, reliable, and useful in computing velocities during a flying 20-m sprint, and therefore can be recommended to both coaches and researchers

Sympathomimetic effects of chronic methamphetamine abuse on oral health: a cross-sectional study

Background: Methamphetamine, a highly addictive sympathomimetic stimulant, is currently widely abused worldwide and has been associated with devastating effects on oral health, resulting in the term "meth mouth". However, "meth mouth" pathology is primarily based on case reports with a lack of systematic clinical evaluation. Therefore, we have conducted a systematic study to investigate (1) the pharmacological impact of methamphetamine on oral health with regard to saliva function, including the parameters saliva flow rate and total saliva production (ml/5 min) and the buffering capacity of saliva;(2) the contribution of the symptoms of bruxism and muscle trismus to potential oral health damage. Methods: We assessed the data of 100 chronic methamphetamine abusers and 100 matched-pair comparison participants. Primarily, we conducted an anamnesis with all methamphetamine abusers with regard to saliva dysfunctions, jaw clenching and pain in the temporomandibular joint. Subsequently, in the first part of the clinical enquiry, we tested the saliva flow rate and the total saliva production (ml/5 min) by using the sialometry method and the buffer capacity of saliva by determining the pH-value. In the second part of the clinical enquiry, we evaluated bruxism symptoms with respect to generalized tooth attrition, dentine exposure and visible enamel cracks and examined a potential muscle trismus by measuring the maximal opening of the mouth. Results: The majority of methamphetamine abusers reported a dry mouth (72 %) and jaw clenching (68 %). Almost half of all methamphetamine abusers experienced pain in the temporomandibular joint (47 %). With regard to the clinical findings, methamphetamine abusers showed significantly lower total saliva production (ml/5 min) (p 0.05). Conclusions: The sympathomimetic effects of chronic methamphetamine abuse may lead to dry mouth and extensive bruxism and therefore can increase the risk for caries decay, periodontal lesions and tooth wear. Furthermore, a significant decline of saliva buffer capacity in methamphetamine abusers may trigger the risk for dental erosions. Methamphetamine abusers and practitioners should be aware of these symptoms

Caveolin-1-Enhanced Motility and Focal Adhesion Turnover Require Tyrosine-14 but Not Accumulation to the Rear in Metastatic Cancer Cells

Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells

Quantifizierung von Strukturen, Kinetik und Dynamik nekrobiologischer Prozesse mit Hilfe bildverarbeitender konfokaler Fluoreszenzmikroskopie

Translocation of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet is presently discussed as a hallmark of apoptosis. The signalling which initialises the process and the translocation mechanism itself are still under investigation. Diffusion or enzymatic transport is suspected. Staurosporine induced PS-translocation was monitored in HCE-cells and in mouse lymphocytes (EL-4) by Annexin V-FITC (AV) fluorescence. QFM could not only reveal the percentage of AV positive and AV negative cells in both cultures, it could also derive that once initiated, PS-translocation in single HCE-cells is completed within less than ½h. This and other observations support nondiffusive PS-translocation. For toxicological investigations, early apoptotic parameters are essential. QFM detected two very early staurosporine induced effects in HCE- and EL-4-cells. In HCE-cells, a morphologic form of Karyorrhexis was discovered as early as 10min after induction. This form of chromatin condensation is probably initiated by the mitochondrial apoptotic inducing factor (AIF), which was recently discovered. Activated caspase-3, which is also discussed to initialise PS-translocation and chromatin condensation could be excluded for both events in HCE-cells. In EL-4-cells, perturbation of cellular migration and the exclusion of small cellular vesicles were detected within minutes after staurosporine induction. The results presented in this and other scientific publications validate the OFM method as a promising tool for future investigations. The method should work on many cellular events, in which fluorescent or non fluorescent texture or morphological features must be connected with statistical information

Quantifying of structures, kinetics and dynamics of necrobiological processes by image processed confocal fluorescence microscopy

Translocation of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet is presently discussed as a hallmark of apoptosis. The signalling which initialises the process and the translocation mechanism itself are still under investigation. Diffusion or enzymatic transport is suspected. Staurosporine induced PS-translocation was monitored in HCE-cells and in mouse lymphocytes (EL-4) by Annexin V-FITC (AV) fluorescence. QFM could not only reveal the percentage of AV positive and AV negative cells in both cultures, it could also derive that once initiated, PS-translocation in single HCE-cells is completed within less than ½h. This and other observations support nondiffusive PS-translocation. For toxicological investigations, early apoptotic parameters are essential. QFM detected two very early staurosporine induced effects in HCE- and EL-4-cells. In HCE-cells, a morphologic form of Karyorrhexis was discovered as early as 10min after induction. This form of chromatin condensation is probably initiated by the mitochondrial apoptotic inducing factor (AIF), which was recently discovered. Activated caspase-3, which is also discussed to initialise PS-translocation and chromatin condensation could be excluded for both events in HCE-cells. In EL-4-cells, perturbation of cellular migration and the exclusion of small cellular vesicles were detected within minutes after staurosporine induction. The results presented in this and other scientific publications validate the OFM method as a promising tool for future investigations. The method should work on many cellular events, in which fluorescent or non fluorescent texture or morphological features must be connected with statistical information

Location matters: the endoplasmic reticulum and protein trafficking in dendrites

Neurons are highly polarized, but the trafficking mechanisms that operate in these cells and the topological organization of their secretory organelles are still poorly understood. Particularly incipient is our knowledge of the role of the neuronal endoplasmic reticulum. Here we review the current understanding of the endoplasmic reticulum in neurons, its structure, composition, dendritic distribution and dynamics. We also focus on the trafficking of proteins through the dendritic endoplasmic reticulum, emphasizing the relevance of transport, retention, assembly of multi-subunit protein complexes and export. We additionally discuss the roles of the dendritic endoplasmic reticulum in synaptic plasticity

Shape Transitions and Lattice Structuring of Ceramide-Enriched Domains Generated by Sphingomyelinase in Lipid Monolayers

Sphingomyelinases (SMases) hydrolyze the membrane constituent sphingomyelin (SM) to phosphocholine and ceramide (Cer). Growing evidence supports that SMase-induced SM→Cer conversion leads to the formation of lateral Cer-enriched domains which drive structural reorganization in lipid membranes. We previously provided visual evidence in real-time for the formation of Cer-enriched domains in SM monolayers through the action of the neutral Bacillus cereus SMase. In this work, we disclose a succession of discrete morphologic transitions and lateral organization of Cer-enriched domains that underlay the SMase-generated surface topography. We further reveal how these structural parameters couple to the generation of two-dimensional electrostatic fields, based upon the specific orientation of the lipid dipole moments in the Cer-enriched domains. Advanced image processing routines in combination with time-resolved epifluorescence microscopy on Langmuir monolayers revealed: 1), spontaneous nucleation and circular growth of Cer-enriched domains after injection of SMase into the subphase of the SM monolayer; 2), domain-intrinsic discrete transitions from circular to periodically undulating shapes followed by a second transition toward increasingly branched morphologies; 3), lateral superstructure organization into predominantly hexagonal domain lattices; 4), formation of super-superstructures by the hexagonal lattices; and 5), rotationally and laterally coupled domain movement before domain border contact. All patterns proved to be specific for the SMase-driven system since they could not be observed with Cer-enriched domains generated by defined mixtures of SM/Cer in enzyme-free monolayers at the same surface pressure (Π = 10 mN/m). Following the theories of lateral shape transitions, dipolar electrostatic interactions of lipid domains, and direct determinations of the monolayer dipole potential, our data show that SMase induces a domain-specific packing and orientation of the molecular dipole moments perpendicular to the air/water interface. In consequence, protein-driven generation of specific out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-specific out-of-equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (μm) scales. The cross-talk between lateral domain structures and dipolar electrostatic fields adds new perspectives to the mechanisms of SMase-mediated signal transduction in biological membranes