Fluorescence correlation spectroscopy measurements on amyloid fibril reveal at least two binding modes for fluorescent sensors

Interaction of fluorophore with amyloid structure is central to the design and development of fluorescent sensors. In literature ensemble spectroscopy results reveal at least two distinct binding modes for most of the torsional probes like Thioflavin-T, SYPRO Orange, etc. though single molecule localization microscopy and spectroscopy results consider only one binding mode. To address this discrepancy we employed fluorescence correlation spectroscopy (FCS) to measure the binding kinetics on immobilized fibril structures with single molecule sensitivity. Our results indicate that there are at least two possible biding configurations with differing relaxation time constants indicating possible weaker and stronger binding configurations depending on the local environment on the fibril structure. Critical evaluation of on-time distributions from the recorded wide-field image stack for single molecule localization microscopy image, further highlight the presence of two burst times. Such bi-modal binding behavior is attributed to the differing binding configurations on fibril architecture

The tunnel wall collapse and pothole creation on the hilly terrain surface: a case study of stabilization

An approximately 2-km-long tunnel is located in the heart of the Bailadila hills in India and is used for the transportation of iron ore excavated from nearby mines. A portion measuring 2 m height and 5 m width of the Reinforced Cement Concrete (RCC) wall of this tunnel was severely ruptured and caved in bringing a lot of debris and the slush. This caused subsidence on the hilly terrain surface exactly overlying the affected tunnel portion. Various stabilization measures consisting of cement grouting in tandem with chemicals were undertaken to strengthen the failed concrete lining as well as the rock and soil mass of tunnel walls and roof in and around the collapsed portion. Additional stabilization measures were also undertaken to back-fill the cavity/pothole formed on the hilly terrain surface. Further, monitoring of the stabilized zone was carried out by installing mechanical type convergence indicators in the tunnel walls and roof. Also, the efficacy of grout was estimated using an ultrasound sonic tester and through Schmidt hammer rebound tests. The paper presents this case study along with the conclusions and recommendations, which were drawn based on this study

Photon Antibunching Reveals Static and Dynamic Quenching Interaction of Tryptophan with Atto-655

Fluorescence correlation spectroscopy (FCS) of photoinduced electron transfer (PET) between the dye Atto-655 and the amino acid tryptophan has been extensively used for studying fast conformational dynamics of small disordered peptides and proteins. However, a precise understanding of the quenching mechanism and its exact rates that would explain ensemble as well as single-molecule spectroscopy results is still lacking. In this contribution, a general unified model for intermolecular PET between Atto-655 and tryptophan is developed, which involves ground-state complex formation, quenching sphere of action, and dynamic quenching at the single-molecule level. We present measurements of fluorescence antibunching, fluorescence lifetime, and steady-state fluorescence intensity and absorbance and demonstrate that our model is capable to describe all results in a global and coherent manner

Single-molecule detection in exploring nanoenvironments: an overview

In the last one decade or so, a variety of optical experiments have been designed and performed that are capable of exploring down to the regime of single-molecule detection and measurements in all different environments, including solids, surfaces, and liquids. Single-molecule detection in condensed phases has many important chemical and biological applications. A few to list are: rapid DNA sequencing, DNA fragment sizing, medical diagnosis, forensic analysis, understanding of chemical dynamics and mechanisms, etc. Single-molecule spectroscopy allows us to observe the individual molecules hidden in a condensed phase sample, by using a tunable laser light. This technique has the ability to detect and monitor systems with an ultimate sensitivity level of ~1.66 × 10-24 moles (1/N0). Measurement at the single-molecule level can completely remove the complicacy associated with ensemble-averaged macroscopic measurements. It allows us to construct a frequency histogram of the distribution of values for a parameter of interest following a large number of measurements on many individual molecules. Such a distribution carries much more information than the average value of the parameter obtained from a macroscopic measurement. As there is no ensemble averaging involved, only measurements at the single-molecule level can give an appropriate test for microscopic dynamical theories. Using single-molecule spectroscopy one can, in principle, follow the temporal evolution of any complex reaction path. As the field is still emerging, with newer methodologies of detecting single molecules with improved signal-to-noise ratios, it is expected that many new physical and chemical phenomena will certainly be explored using this technique. In the present article, our endeavor is to give an overview of the different aspects of single-molecule detection, along with some of its important applications in the areas of bioscience and chemical physics