Precise Particle Tracking Against a Complicated Background: Polynomial Fitting with Gaussian Weight

We present a new particle tracking software algorithm designed to accurately track the motion of low-contrast particles against a background with large variations in light levels. The method is based on a polynomial fit of the intensity around each feature point, weighted by a Gaussian function of the distance from the centre, and is especially suitable for tracking endogeneous particles in the cell, imaged with bright field, phase contrast or fluorescence optical microscopy. Furthermore, the method can simultaneously track particles of all different sizes, and allows significant freedom in their shape. The algorithm is evaluated using the quantitative measures of accuracy and precision of previous authors, using simulated images at variable signal-to-noise ratios. To these we add a new test of the error due to a non-uniform background. Finally the tracking of particles in real cell images is demonstrated. The method is made freely available for non-commencial use as a software package with a graphical user-inferface, which can be run within the Matlab programming environment

Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy

The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution

Ranolazine in Symptomatic Diabetic Patients Without Obstructive Coronary Artery Disease: Impact on Microvascular and Diastolic Function

Background: Treatments for patients with myocardial ischemia in the absence of angiographic obstructive coronary artery disease are limited. In these patients, particularly those with diabetes mellitus, diffuse coronary atherosclerosis and microvascular dysfunction is a common phenotype and may be accompanied by diastolic dysfunction. Our primary aim was to determine whether ranolazine would quantitatively improve exercise‐stimulated myocardial blood flow and cardiac function in symptomatic diabetic patients without obstructive coronary artery disease. Methods and Results: We conducted a double‐blinded crossover trial with 1:1 random allocation to the order of ranolazine and placebo. At baseline and after each 4‐week treatment arm, left ventricular myocardial blood flow and coronary flow reserve (CFR; primary end point) were measured at rest and after supine bicycle exercise using 13N‐ammonia myocardial perfusion positron emission tomography. Resting echocardiography was also performed. Multilevel mixed‐effects linear regression was used to determine treatment effects. Thirty‐five patients met criteria for inclusion. Ranolazine did not significantly alter rest or postexercise left ventricular myocardial blood flow or CFR. However, patients with lower baseline CFR were more likely to experience improvement in CFR with ranolazine (r=−0.401, P=0.02) than with placebo (r=−0.188, P=0.28). In addition, ranolazine was associated with an improvement in E/septal e′ (P=0.001) and E/lateral e′ (P=0.01). Conclusions: In symptomatic diabetic patients without obstructive coronary artery disease, ranolazine did not change exercise‐stimulated myocardial blood flow or CFR but did modestly improve diastolic function. Patients with more severe baseline impairment in CFR may derive more benefit from ranolazine. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT01754259

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Quantitative comparison of algorithms for tracking single fluorescent particles.

Single particle tracking has seen numerous applications in biophysics, ranging from the diffusion of proteins in cell membranes to the movement of molecular motors. A plethora of computer algorithms have been developed to monitor the sub-pixel displacement of fluorescent objects between successive video frames, and some have been claimed to have "nanometer" resolution. To date, there has been no rigorous comparison of these algorithms under realistic conditions. In this paper, we quantitatively compare specific implementations of four commonly used tracking algorithms: cross-correlation, sum-absolute difference, centroid, and direct Gaussian fit. Images of fluorescent objects ranging in size from point sources to 5 microm were computer generated with known sub-pixel displacements. Realistic noise was added and the above four algorithms were compared for accuracy and precision. We found that cross-correlation is the most accurate algorithm for large particles. However, for point sources, direct Gaussian fit to the intensity distribution is the superior algorithm in terms of both accuracy and precision, and is the most robust at low signal-to-noise. Most significantly, all four algorithms fail as the signal-to-noise ratio approaches 4. We judge direct Gaussian fit to be the best algorithm when tracking single fluorophores, where the signal-to-noise is frequently near 4

Management of Coronary Artery Calcium and Coronary CTA Findings

Coronary artery calcium (CAC) testing and coronary computed tomography angiography (CTA) have significant data supporting their ability to identify coronary artery disease (CAD) and classify patient risk for atherosclerotic cardiovascular disease (ASCVD). Evidence regarding CAC use for screening has established an excellent prognosis in patients with no detectable CAC, and the ability to risk re-classify the majority of asymptomatic patients considered intermediate risk by traditional risk scores. While data regarding the ideal management of CAC findings are limited, evidence supports statin consideration in patients with CAC > 0 and individualized aspirin therapy accounting for CAD risk factors, CAC severity, and factors which increase a patient’s risk of bleeding. In patients with stable or acute symptoms undergoing coronary CTA, a normal CTA predicts excellent prognosis, allowing reassurance and disposition without further testing. When CTA identifies nonobstructive CAD (<50 % stenosis), observational data support consideration of statin use/intensification in patients with extensive plaque (at least four coronary segments involved) and patients with high-risk plaque features. In patients with both nonobstructive and obstructive CAD, multiple studies have now demonstrated an ability of CTA to guide management and improve CAD risk factor control. Still, significant under-treatment of cardiovascular risk factors and high-risk image findings remain, among concerns that CTA may increase invasive angiography and revascularization. To fully realize the impact of atherosclerosis imaging for ASCVD prevention, patient engagement in lifestyle changes and the modification of ASCVD risk factors remain the foundation of care. This review provides an overview of available data and recommendations in the management of CAC and CTA findings

Image Analysis with Rapid and Accurate Two-Dimensional Gaussian Fitting

A computationally rapid image analysis method, weighted overdetermined regression, is presented for two-dimensional (2D) Gaussian Fitting of particle location with subpixel resolution from a pixelized image of light intensity. Compared to least-squares Gaussian iterative fitting, which is most exact but prohibitively slow for large data sets, the precision of this new method is equivalent when the signal-to-noise ratio is high and approaches it when the signal-to-noise ratio is low, while enjoying a more than 100-fold improvement in computational time. Compared to another widely used approximation method, nine-point regression, we show that precision and speed are both improved. Additionally, weighted regression runs nearly as fast and with greatly improved precision compared to the simplest method, the moment method, which, despite its limited precision, is frequently employed because of its speed. Quantitative comparisons are presented for both circular and elliptical Gaussian intensity distributions. This new image analysis method may be useful when dealing with large data sets such as those frequently met in astronomy or in single-particle and single-molecule tracking using microscopy and may facilitate advances such as real-time quantification of microscopy images