Dynamic label-free imaging of lipid nanodomains

Lipid rafts are submicron proteolipid domains thought to be responsible for membrane trafficking and signaling. Their small size and transient nature put an understanding of their dynamics beyond the reach of existing techniques, leading to much contention as to their exact role. Here, we exploit the differences in light scattering from lipid bilayer phases to achieve dynamic imaging of nanoscopic lipid domains without any labels. Using phase-separated droplet interface bilayers we resolve the diffusion of domains as small as 50 nm in radius and observe nanodomain formation, destruction, and dynamic coalescence with a domain lifetime of 220 ± 60 ms. Domain dynamics on this timescale suggests an important role in modulating membrane protein function.Cell membranes compartmentalize into lipid domains that enable the selective recruitment of specific proteins (1). These “lipid rafts” have been proposed to control many membrane processes including apical sorting, protein trafficking, and the clustering of proteins during signaling. The dynamic formation and destruction of lipid nanodomains are thought to provide the central mechanism to regulate this wide range of essential processes (2–4). Although many methods now provide strong evidence to support their existence in vivo (5), the combination of nanoscopic size and dynamics on millisecond timescales has placed the direct observation of their behavior beyond the scope of existing techniques. Consequently, although we know they exist, frustratingly little is known regarding their function and dynamics (6).Recent advances in fluorescence nanoscopy provide the only time-dependent information on the behavior of lipid nanodomains (7–9). Stimulated emission depletion–fluorescence correlation spectroscopy has shown cholesterol-mediated hindered nanoscale diffusion of single labeled sphingomyelin lipids that is consistent with the lipid raft hypothesis and transient binding of lipids (9). Superresolution fluorescence microscopy has also revealed protein clusters in cell membranes with 0.5-s temporal resolution (7). All of these experiments, however, are limited in temporal resolution by fluorescence, and must infer lipid nanodomains from the addition of fluorescent labels.Macroscopic phase separation in artificial lipid bilayers has been widely used to help understand the biological implications of domain formation. Different lipid phases can be visualized using fluorescence microscopy with labels that preferentially partition into a specific phase (10–12). This approach is successful for micrometer-sized domains but inevitably fails on the tens to few hundreds of nanometers scale due to limitations in phase specificity, the limited residence time of a label within a specific nanoscopic domain, and the achievable optical resolution (13). The fluorescent probe is itself an additional component that can perturb phase behavior, either directly or through photooxidation (14, 15). As a result, lipid nanodomain dynamics have not been observed directly even in artificial systems, although recent ensemble-based techniques report lipid heterogeneity on the appropriate length scales (13). In addition to fluorescence-based approaches, ellipsometry and reflection interference contrast microscopy have been used to characterize phase separation in lipid bilayers (16, 17), taking advantage of different bilayer thicknesses and refractive indices caused by varying degrees of cholesterol content and lipid packing. Given sufficient sensitivity and resolution, this approach should hold for arbitrarily small domains.We recently developed interferometric scattering microscopy (iSCAT) (18–20) and achieved sensitivity to refractive index perturbations down to the level of a single unlabeled protein molecule in solution with millisecond time resolution (21, 22). Here, we exploit the unique sensitivity of iSCAT to overcome the limitations in temporal resolution and sensitivity to image, track, and characterize lipid nanodomains without requiring any labels. We use droplet interface bilayers (DIBs) as an artificial membrane model (23, 24) with phase-separated lipid mixtures (Fig. 1A). DIBs are formed by the contact of two lipid monolayers; in this case, a monolayer formed at the interface between an aqueous droplet and a solution of phospholipids in oil, and another between a thin hydrogel film and the oil. DIBs are robust, long-lived, and defect-free, show unrestricted diffusion, form gigaohm resistance seals, and are compatible with high-resolution optical microscopy (24).Open in a separate windowFig. 1.Detection of lipid nanodomains using iSCAT. (A) Schematic of a DIB showing ordered (light gray) and disordered (black) phases. The interference between scattered and reflected fields (E_s and E_r) is detected in the far field using a digital camera. (B) The 100-ms TIRF (Top) and iSCAT (Bottom) images of a DIB containing S_o domains within a bulk L_d phase (1:1 DOPC: bSM plus 1 mol% Atto488-DPPE). The static background due to scattering from the agarose substrate can been seen in this raw iSCAT image. This background is subtracted in subsequent images. (C) Time-lapse sequence of iSCAT images of L_o nanodomains appearing from a uniform L_d phase upon cooling of a DIB below the phase transition temperature. The droplet was heated to 45 °C for 10 min. Nanodomains appeared 2–5 min after heating was stopped. Composition, 1:1:1 DPhPC:bSM:Chol. Greyscale values are of the normalized reflected intensity. (D) Trajectories corresponding to average pixel contrast within a 900 × 900-nm window centered on each nanodomain shown in C. Values before the appearance of the domain are representative of the background fluctuations at the position where the domain first becomes visible. (Scale bars: 5 μm.)     

Aortic valve calcification: its significance and limitation as a marker for coronary artery disease

Aortic valve calcification (AVC) is correlated with atherosclerotic risk factors; however, its significance remains largely unknown. The aim of this study was to investigate whether AVC detected by transthoracic echocardiography can be a useful marker for the identification of significant coronary artery disease (CAD), particularly in elderly patients. The study included 432 consecutive patients with suspected CAD who were admitted for the first time for coronary angiography. Two-dimensional transthoracic echocardiography and selective coronary angiography were performed in all patients. Aortic valve calcification was defined as bright dense echoes of > 1 mm on one or more cusps and decreased mobility of the involved cusp. Aortic valve calcification was detected in 64 of the 337 patients with significant CAD, but only in 9 of 95 cases with normal or mildly stenotic coronary arteries (19% vs 9%, p < 0.001). The severity of coronary artery disease (defined as the number of obstructed vessels) was not related to the presence of AVC (p > 0.05). Stepwise multiple logistic regression analysis of the study patients revealed only age (p=0.003, odds ratio= 1.56) and AVC (p<0.001, odds ratio = 2.03) as independent predictors of CAD. When the study population was divided into two groups as those below (n = 338) and above (n = 94) 75 years old, AVC failed to be a predictor of CAD in those >75 years old (p > 0.05, odds ratio = 0.8) while it remained the most significant predictor of CAD (p<0.001, odds ratio=2.19 in patients aged <75 years. In conclusion, detection of AVC by transthoracic echocardiography may be a useful noninvasive marker for identification of significant CAD in patients younger than 75 years old. Its clinical usefulness is limited in elderly patients.     

Comparison and evaluation of experimental mediastinitis models: precolonized foreign body implants and bacterial suspension inoculation seems promising

Background  

Post-sternotomy mediastinitis (PSM) is a devastating surgical complication affecting 1–3% of patients that undergo cardiac surgery. Staphylococcus aureus is one of the most commonly encountered bacterial pathogen cultured from mediastinal samples obtained from patients with PSM. A component of the membrane of the gram positive bacteria, lipoteichoic acid, stimulates the blood monocytes and macrophages to secrete cytokines, radicals and nitrogen species leading to oxido-inflammatory damage. This seems to be responsible for the high mortality rate in PSM. For the evaluation of the pathogenesis of infection or for the investigation of alternative treatment models in infection, no standard model of mediastinitis seems to be available. In this study, we evaluated four mediastinitis models in rats.     

Endovascular Stents in Arterial Injury after Radiotherapy

We evaluated preliminary success and patency of stenting for the treatment of radiation-induced arterial diseases. Thirteen stents were placed in 8 patients to treat occlusion (n=3), aneurysm (n=1), residual stenosis (n=2),multiple stenoses (n=1), and delayed restenosis after previous balloon angioplasty (n=1). Interventional procedure was successfully performed in 8 patients for their arterial lesions after radiotherapy. Six patients underwent interventional procedure once or twice. Two patients underwent PTA 4 times. Five of these patients demonstrated primary patency with relief of clinical symptoms with a mean follow-up of 2 years (range: 8-60 months). Clinical improvement was noted in the other patients. Our results suggest that stent placement by single or multiple techniques may have immediate effect on arterial lesions caused by radiation and can be considered as a therapeutic option of choice in these cases.     

Are occult pneumothoraces truly occult or simply missed?

BACKGROUND: Nonradiologists typically diagnose pneumothoraces (PTX) based on a visible pleural stripe. PTXs not seen on supine AP chest radiographs (CXR), but appreciated on a computed tomographic (CT) scan, termed occult pneumothoraces (OPTX), are increasingly common. The purpose was to (1) determine whether perceived OPTXs were truly occult or simply missed and (2) address factors that contribute to the poor sensitivity of the supine CXR. METHODS: A previous study of severely injured patients (ISS >or =12) identified 44 patients with OPTXs. JPEG images of these CXRs were randomly arranged with images of 11 injured patients without PTXs (CT proven). Three unique groups of radiologists reviewed the images for signs of PTXs, and determined if a thoracic CT was subsequently required. RESULTS: Retrospective review identified only 12 to 24% of the OPTXs depending on radiology group. The kappa inter-observer agreement value was 0.55 to 0.56 (poor agreement). PTXs were most commonly identified via the deep sulcus sign (75-90%). CXRs were considered inadequate in 16 to 25% of OPTX images and in 0 to 18% of images without OPTXs. Thoracic CT scans were recommended in 18 to 33% of patients with inadequate CXRs, but 67 to 82% of patients with adequate CXRs. CONCLUSIONS: Less than 24% of all OPTXs might have been inferred from subtle radiologic findings, such as the deep sulcus sign. The majority of OPTX cases (50-64%) did not warrant a CT scan based on other findings. Concern for an OPTX after severe trauma is a valid indication for thoracic CT.