Labeling fibrin fibers with microparticle sized beads alter single fibrin fiber lysis, external clot lysis, and produce large fibrin aggregates upon lysis

Background Fluorescent beads are often used as a tool for visualizing fibrin fibers and can mimic the size of microparticles in the blood. Studies showed microparticles alter the appearance and behavior of whole blood clot systems. Objectives Here we investigate the effect of beads on fibrin fiber lysis and extensibility to enhance understanding of this common research technique and as a biomimetic system for fibrin-microparticle interaction. Methods We used fluorescence microscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM) to quantify changes in lysis, extensibility, and clot structure of fibrin fibers and clots in the presence and absence of beads. Results and Conclusions Fibrin clot structure and lysis were altered in the presence of beads. Fibrin clots formed with beads had a higher fiber density, smaller fibers, and smaller pores. The rate of lysis for clots was reduced when beads were present. Lysis of bead-labeled individual fibers showed that beads, at concentrations similar to those reported for microparticles in the blood, cause a subset of fibers to resist lysis. In the absence of beads, all fibers lyse. These results demonstrate that beads alter fiber lysis through both a change in fibrin clot structure as well as changes to individual fiber lysis behavior. Additionally, the lysis of clots with beads produced large fibrin aggregates. This data encourages researchers to use careful consideration when labeling fibrin fibers with fluorescent beads and suggests that particles binding fibrin(ogen) in the bloodstream may be an underappreciated mechanism increasing the risk of thrombosis.

Measurement of the Concentration and the Brightness for Samples Containing Multiple Molecules with Different Brightness Using Fluorescence Correlation Spectroscopy

In this study, the concentration and brightness measured by fluorescence correlation spectroscopy (FCS) in samples containing multiple species with different brightness levels was demonstrated. FCS measurements of such samples are generally difficult. However, the calculation we introduced here can provide the measurement results of the FCS. The effectiveness of the calculation was investigated based on simulations and experiments in the case of a mixture of fluorescent beads with known brightness and other fluorescent beads with unknown brightness. The results show that the concentration of the known brightness agrees well with the expected values. The obtained concentration and brightness of the species with unknown brightness is possible, and it worked well in the simulation; however, the accuracy for the species was lower than that of the species with known brightness. As a result, the calculation is useful in measuring the concentration of species with known brightness in samples containing undesired bright species, such as aggregation. The calculation for the species with unknown brightness may also be useful if good protocols or instruments are established in the future.

Hyperspectral Three-Dimensional Fluorescence Imaging Using Snapshot Optical Tomography

Hyperspectral three-dimensional (3D) imaging can provide both 3D structural and functional information of a specimen. The imaging throughput is typically very low due to the requirement of scanning mechanisms for different depths and wavelengths. Here we demonstrate hyperspectral 3D imaging using Snapshot projection optical tomography (SPOT) and Fourier-transform spectroscopy (FTS). SPOT allows us to instantaneously acquire the projection images corresponding to different viewing angles, while FTS allows us to perform hyperspectral imaging at high spectral resolution. Using fluorescent beads and sunflower pollens, we demonstrate the imaging performance of the developed system.

Preparation of fluorescent beads under C. elegans for fluorescence microscopy with adaptive optics

We describe a protocol for the preparation of a sample of fluorescent beads under C. elegans. This type of sample is useful for research into the use of adaptive optics to correct sample-induced optical aberrations in fluorescence microscopy.

Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.

A24 FCGBP MAINTAINS MUC2 MUCUS STRUCTURAL INTEGRITY BY STABILIZING THE MUCUS LAYER IN RESPONSE TO THE COLONIC PATHOGEN, ENTAMOEBA HISTOLYTICA

Background MUC2 mucin is the major component of the colonic mucus bilayer that serves as the first line of innate host defense against pathogens while supporting a healthy microbiota and regulating epithelial barrier function. Proteomic studies of colonic mucus have identified various mucus-associated proteins. One of the most abundant is FCGBP, similar to MUC2 mucin, but its interaction with MUC2 or function is not known. Here, we elucidated FCGBP functional role in stabilizing MUC2 mucus and in innate host defence against Entamoeba histolytica (Eh). Aims Hypothesis: MUC2 mucin and FCGBP are coordinately produced and play an important role in innate host defense. The specific aims are: 1. To determine if FCGBP alters the structural integrity of the mucus layer 2. To determine the role of FCGBP in Eh infection Methods FCGBP mRNA and protein expression induced by Eh, in WT and FCGBP CRISPR/Cas9 LS174T goblet cells were analysed by RT-PCR and Western blotting. To compare integrity of the mucus layer, fluorescent Eh and 1μM fluorescent beads were inoculated on WT and KO monolayers and adherent Eh and bead penetrability analyzed. To quantify MUC2 and FCGBP degradation by Eh, purified MUC2 and recombinant FCGBP were incubated with Eh proteases (SPs) and Western blotted using highly specific antibodies against various regions of the proteins. Results In response to live Eh, FCGBP and MUC2 mRNA and protein expressions were significantly increased in a time-dependent manner. Surprisingly, FCGBP KO cells elicited robust expression of pro-inflammatory cytokine mRNA and protein as compared to WT cells. More fluorescent Eh were attached to the mucus layer of FCGBP KO cells as compared to WT or MUC2 KO cells. Fluorescent beads penetrated further towards the epithelial cell surface in KO as compared to WT cells. Interestingly, while both MUC2 and FCGBP from purified polymeric mucins were degraded by Eh SPs, FCGBP cleavage occurred at a faster rate than MUC2. Degradation of FCGBP and MUC2 was mediated by EhCP-A5 cysteine proteinase using purified MUC2 and recombinant FCGBP. Conclusions In WT goblet cells, FCGBP and MUC2 were upregulated temporally in response to Eh. The increase in pro-inflammatory cytokine expression in FCGBP KO cells in response to Eh suggests that Eh directly interacted with the cell surface suggesting an impaired protective mucus layer. In support of this, fluorescent beads penetrated the mucus layer close to the cell surface and more Eh were attached to FCGBP KO mucus demonstrating that FCGBP was critical in providing structural integrity of the mucus layer. In response to Eh, FCGBP degradation was a prerequisite for MUC2 cleavage, providing direct evidence that FCGBP and MUC2 interactions conferred biophysical properties of the protective functions of the mucus gel. Funding Agencies CIHR