Sensitive Fluorescence Detection using a Camera from the Gaming Industry

The detection limit for fluorescence imaging has been improved by more than two orders of magnitude by a modulation technique that minimizes the influence of noise. Since most noise sources have a 1/f dependence, higher modulation frequencies result in less noise in the detected signal. Typical cameras used for imaging have frame rates of 50 or 100 hertz which is too low for substantial noise reduction. However, new time-of-flight cameras developed for the gaming industry have modulation frequencies of up to 20 MHz, allowing for a substantial reduction in noise in the detected signal. In this study, the improved detection limit of this camera is quantified and used to image mouse tumors labeled with fluorescent dye. The results of the calibration show that the detection limit is increased by a factor of more than 400 when compared to the non-modulated signal from the camera

Stay calm and focus on the learning outcomes: Tools for taking biophysical chemistry online

Course specific learning outcomes are an important tool to define the scope of a course and can be very helpful when designing experiments and assessments. With slight modification, these learning outcomes can serve as a guide when transitioning to the distance learning format especially in courses with a traditional lab. Here we present such an example for the biophysical chemistry course

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules, and even long microtubules have measured persistence lengths which vary by an order of magnitude. Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL. This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well

The Volatile Anesthetic Isoflurane Increases Endothelial Adenosine Generation via Microparticle Ecto-5′-Nucleotidase (CD73) Release

Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5′-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation

Understanding the Vibrational Features of Hydrogen-Bonded Dimers

Hydrogen-bonded systems often exhibit very broad and unusually shaped features in their vibrational spectra. The origin of these features is oftentimes unclear. Consequently, computational methods are frequently used to model these features in order to better understand their origin. However, reproducing these features with computational methods is often quite challenging. Many methods have been developed each of which has its own advantages and disadvantages. The research presented in this thesis focuses on the development and application of new computational methods to reproduce the multi-hump (broad peak) vibrational features found in many hydrogen-bonded dimers. These methods utilize density functional theory to calculate the potential energy surface and a quantum variational approach to calculate vibrational spectra from these surfaces. One of the methods developed involves adiabatically separating lower frequency vibrational modes, which modulate the hydrogen bond length, from higher frequency modes that contribute to the structure. Another method that was developed uses a classical molecular dynamics simulation, in place of low-frequency modes, to more accurately sample configurations. The results of the calculations performed with these methods indicate that the broadness of these multi-hump features originate from low-frequency modes modulating the hydrogen bond length, while the multi-hump lineshape is derived from strong Fermi resonances between the OH stretch and the OH bending modes

A Combined Electronic Structure and Molecular Dynamics Approach to Computing the OH Vibrational Feature of Strongly Hydrogen-Bonded Carboxylic Acids

Medium and strong hydrogen bonds give rise to vibrational features that can span several hundreds of wavenumbers and have unusual line shapes. For example, dimers consisting of carboxylic acids hydrogen-bonded to nitrogen-containing aromatic bases exhibit a vibrational feature that spans over 900 cm−1and contains two very broad peaks. In this report, we demonstrate how this feature can be reproduced using a combined molecular dynamics (MD) and electronic structure “spectral map” approach, which has been very successful in modeling the vibrational spectrum of water in different environments. In this approach, spectral maps are created, relating the transition frequencies and probabilities to the electric field along the OH bond, which are obtained from the density functional theory calculations of snapshots taken from a classical MD simulation. This map was used to compute the spectral properties of thousands of geometries of the pyridine-acetic acid dimer sampled by a MD simulation, which were used to compute the overall spectral feature. It was found that this approach reproduced the experimental spectrum better than the previous dimer stretch approaches (which were based on describing the dimer geometries harmonically) through a more accurate sampling of dimer geometries. The broadness of these vibrational features largely originates from the range of geometries present in the condensed phase, while the unusual line shape is caused by strong Fermi resonances