Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the "colocalization" of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software.

A two-dimensional multiscale model is introduced for studying formation of a thrombus (clot) in a blood vessel. It involves components for modelling viscous, incompressible blood plasma; non-activated and activated platelets; blood cells; activating chemicals; fibrinogen; and vessel walls and their interactions. The macroscale dynamics of the blood flow is described by the continuum Navier-Stokes equations. The microscale interactions between the activated platelets, the platelets and fibrinogen and the platelets and vessel wall are described through an extended stochastic discrete cellular Potts model. The model is tested for robustness with respect to fluctuations of basic parameters. Simulation results demonstrate the development of an inhomogeneous internal structure of the thrombus, which is confirmed by the preliminary experimental data. We also make predictions about different stages in thrombus development, which can be tested experimentally and suggest specific experiments. Lastly, we demonstrate that the dependence of the thrombus size on the blood flow rate in simulations is close to the one observed experimentally.

A combination of the extended multiscale model, new image processing algorithms, and biological experiments is used for studying the role of Factor VII (FVII) in venous thrombus formation. A detailed submodel of the tissue factor pathway of blood coagulation is introduced within the framework of the multiscale model to provide a detailed description of coagulation cascade. Surface reactions of the extrinsic coagulation pathway on membranes of platelets are studied under different flow conditions. It is shown that low levels of FVII in blood result in a significant delay in thrombin production, demonstrating that FVII plays an active role in promoting thrombus development at an early stage.

An extended multiscale model is introduced for studying the formation of platelet thrombi in blood vessels. The model describes the interplay between viscous, incompressible blood plasma, activated and non-activated platelets, as well as other blood cells, activating chemicals, fibrinogen and vessel walls. The macroscale dynamics of the blood flow is represented by the continuous submodel in the form of the Navier-Stokes equations. The microscale cell-cell interactions are described by the stochastic Cellular Potts Model (CPM). Simulations indicate that increase in flow rates leads to greater structural heterogeneity of the clot. As heterogeneous structural domains within the clot affect thrombus stability, understanding the factors influencing thrombus structure is of significant biomedical importance.

Key Points• CD166 identifies human and murine long-term repopulating stem cells.• CD166 is a functional marker of stem cells and the hematopoietic niche.We previously showed that immature CD166

Thrombus development in mouse mesenteric vessels following laser-induced injury was monitored by high-resolution, near-real-time, two-photon, intravital microscopy. In addition to the use of fluorescently tagged fibrin(ogen) and platelets, plasma was labeled with fluorescently tagged dextran. Because blood cells exclude the dextran in the single plane, blood cells appear as black silhouettes. Thus, in addition to monitoring the accumulation of platelets and fibrin in the thrombus, the protocol detects the movement and incorporation of unlabeled cells in and around it. The developing thrombus perturbs the blood flow near the thrombus surface, which affects the incorporation of platelets and blood cells into the structure. The hemodynamic effects and incorporation of blood cells lead to the development of thrombi with heterogeneous domain structures. Additionally, image processing algorithms and simulations were used to quantify structural features of developing thrombi. This analysis suggests a novel mechanism to stop the growth of developing thrombus.

Abstract-In addition to descriptive biological models, many computational models have been developed for hemostasis/ thrombosis that provide quantitative characterization of thrombus development. Simulations using computational models that have been developed for coagulation reactions, platelet activation, and fibrinogen assembly have been shown to be in close agreement with experimental data. Models of processes involved in hemostasis/thrombosis are being integrated to simulate the development of the thrombus simultaneously in time and space. Key Words: blood coagulation Ⅲ blood flow Ⅲ coagulation Ⅲ platelets Ⅲ thrombosis Ⅲ computational model Ⅲ stochastic multiscale model Ⅲ thrombus development S ignificant progress has been made in our understanding of the hemostatic response. For instance, coagulation pathways 1 have been developed that describe the interactions among different elements and provide insight into the regulation of the response. Similarly, advances in platelet biology 2 have elucidated pathways of platelet activation and identified and characterized molecular components involved in intracellular signaling, as well as surface proteins mediating adhesion to the damaged vessel wall, to other platelets, and to other thrombus components. Furthermore, the development of transgenic, 3,4 gene knockout, and gene knock-in technologies has enabled exploration of the physiological roles of individual components in vivo using sophisticated hemostatic experimental systems. More recently, genomic and proteomic approaches have identified new elements modifying the hemostatic response.The initial identification of hemostatic components and description of coagulation or platelet signaling pathways were qualitative, 4,5 describing the order of interaction among components in coagulation or platelet behavior. These biological and biochemical models were extremely valuable, suggesting how these processes might be regulated and providing an understanding of how deficiencies or dysregulation of particular components leads to pathological states.In addition to these descriptive biological models, computational models have been developed for hemostatic processes that provide quantitative characterization of thrombus development. For instance, the tissue factor (TF)-initiated coagulation model introduced by Hockin et al 6 presented a quantitative description of the network of coagulation reactions. The model correctly predicted that there was a TF concentration threshold required to activate the coagulation system to generate the thrombin required for a hemostatic response. In addition, the computational model introduced by Purvis et al 7 to simulate ADP-mediated platelet activation provided insight into possible mechanisms of negativefeedback signaling and cell-to-cell variation across platelet populations. Furthermore, the kinetic model of fibrin polymerization introduced by Weisel and Nagaswami 8 revealed that changes in the rate of fibrinopeptide cleavage were sufficient to explain many nonintuitive experimental obs...

Introduction Root canal disinfection and the establishment of an intracanal microenvironment conducive to the proliferation/differentiation of stem cells play a significant role in regenerative endodontics. This study was designed to (1) investigate the antimicrobial efficacy of triple antibiotic–containing nanofibers against a dual-species biofilm and (2) evaluate the ability of dental pulp stem cells (DPSCs) to adhere to and proliferate on dentin upon nanofiber exposure. Methods Seven-day-old dual-species biofilm established on dentin specimens was exposed for 3 days to the following: saline (control), antibiotic-free nanofibers (control), and triple antibiotic–containing nanofibers or a saturated triple antibiotic paste (TAP) solution (50 mg/mL in phosphate buffer solution). Bacterial viability was assessed using the LIVE/DEAD assay (Molecular Probes, Inc, Eugene, OR) and confocal laser scanning microscopy. For cyto-compatibility studies, dentin specimens after nanofiber or TAP (1 g/mL in phosphate buffer solution) exposure were evaluated for cell adhesion and spreading by actin-phalloidin staining. DPSC proliferation was assessed on days 1, 3, and 7. Statistics were performed, and significance was set at the 5% level. Results Confocal laser scanning microscopy showed significant bacterial death upon antibiotic-containing nanofiber exposure, differing significantly (P < .05) from antibiotic-free fibers and the control (saline). DPSCs showed enhanced adhesion/spreading on dentin specimens treated with antibiotic-containing nanofibers when compared with its TAP counterparts. The DPSC proliferation rate was similar on days 1 and 3 in antibiotic-free nanofibers, triple antibiotic–containing nanofibers, and TAP-treated dentin. Proliferation was higher (9-fold) on dentin treated with antibiotic-containing nanofibers on day 7 compared with TAP. Conclusions Triple antibiotic–containing polymer nanofibers led to significant bacterial death, whereas they did not affect DPSC attachment and proliferation on dentin.