Characterizing the conformational free-energy landscape of RNA using single-molecule field-effect transistors

We have developed and used high-time-resolution, single-molecule field-effect transistors (smFETs) to characterize the con-formational free-energy landscape of RNA stem-loops. Stem-loops are some of the most common RNA structural motifs and serve as building blocks for the formation of more complex RNA structures. Given their prevalence and integral role in RNA folding, the kinetics of stem-loop (un)folding has been extensively characterized using both experimental and computational approaches. Interestingly, these studies have reported vastly disparate timescales of (un)folding, which has been recently in-terpreted as evidence that (un)folding of even simple stem-loops occurs on a highly rugged conformational energy landscape. Because smFETs do not rely on fluorophore reporters of conformation or on the application of mechanical (un)folding forces, they provide a unique and complementary approach that has allowed us to directly monitor tens of thousands of (un)folding events of individual stem-loops at a 200 μs time resolution. Our results show that under our experimental conditions, stem-loops fold and unfold over a 1-200 ms timescale during which they transition between ensembles of unfolded and folded conformations, the latter of which is composed of at least two sub-populations. The 1-200 ms timescale of (un)folding we observe here indicates that smFETs report on complete (un)folding trajectories in which relatively extended unfolded con-formations of the RNA spend long periods of time wandering the free-energy landscape before sampling one of several mis-folded conformations or, alternatively, the natively folded conformation. Our findings demonstrate how the combination of single-molecule sensitivity and high time resolution makes smFETs unique and powerful tools for characterizing the con-formational free-energy landscape of RNA and highlight the extremely rugged landscape on which even the simplest RNA structural elements fold.

A 4π time-of-flight detector for the ND280/T2K upgrade

ND280 is a near detector of the T2K experiment which is located in the J-PARC accelerator complex in Japan. After a decade of fruitful data-taking, ND280 is scheduled for upgrade. The time-of-flight (ToF) detector, which is described in this article, is one of three new detectors that will be installed in the basket of ND280. The ToF detector has a modular structure. Each module represents an array of 20 plastic scintillator bars which are stacked in a plane of 2.4 × 2.2 m2 area. Six modules of similar construction will be assembled in a cube, thus providing an almost 4π enclosure for an active neutrino target and two TPCs. The light emitted by scintillator is absorbed by arrays of large-area silicon photo-multipliers (SiPMs) which are attached to both ends of every bar. The readout of SiPMs, shaping and analog sum of individual SiPM signals within the array are performed by a discrete circuit amplifier. An average time resolution of about 0.14 ns is achieved for a single bar when measured with cosmic muons. The detector will be installed in the basket of ND280, where it will be used to veto particle originating outside the neutrino target, improve the particle identification and provide a cosmic trigger for calibration of detectors which are enclosed inside it.

The CMS electromagnetic calorimeter upgrade: high-rate readout with precise time and energy resolution

The electromagnetic calorimeter (ECAL) of the CMS detector has played an important role in the physics program of the experiment, delivering outstanding performance throughout data taking. The high-luminosity LHC will pose new challenges. The four to five-fold increase of the number of interactions per bunch crossing will require superior time resolution and noise rejection capabilities. For these reasons the electronics readout has been completely redesigned. A dual gain trans-impedance amplifier and an ASIC providing two 160 MHz ADC channels, gain selection, and data compression will be used in the new readout electronics. The trigger decision will be moved off-detector and will be performed by powerful and flexible FPGA processors, allowing for more sophisticated trigger algorithms to be applied. The upgraded ECAL will be capable of high-precision energy measurements throughout HL-LHC and will greatly improve the time resolution for photons and electrons above 10 GeV.

Timing Performance Simulation for 3D 4H-SiC Detector

To meet the high radiation challenge for detectors in future high-energy physics, a novel 3D 4H-SiC detector was investigated. Three-dimensional 4H-SiC detectors could potentially operate in a harsh radiation and room-temperature environment because of its high thermal conductivity and high atomic displacement threshold energy. Its 3D structure, which decouples the thickness and the distance between electrodes, further improves the timing performance and the radiation hardness of the detector. We developed a simulation software—RASER (RAdiation SEmiconductoR)—to simulate the time resolution of planar and 3D 4H-SiC detectors with different parameters and structures, and the reliability of the software was verified by comparing the simulated and measured time-resolution results of the same detector. The rough time resolution of the 3D 4H-SiC detector was estimated, and the simulation parameters could be used as guideline to 3D 4H-SiC detector design and optimization.

Advanced scintillation materials for calorimetry at circular colliders

The most probable scenario for the development of experimental high-energy physics in the next 50 years is the creation of a family of Future Circular Colliders (FCC) at CERN, a Circular Electron–Positron Collider at China, and a Future Electron-Ion Collider at Brookhaven (USA), which continue the Large Hadron Collider (LHC) scientific program within the framework of the Standard Model and beyond it. The first generation of colliders to be put into operation will utilize the electron beam as one of the colliding species to provide precise mass spectroscopy in a wide energy range. Similarly to the measurements at the high luminosity phase of the LHC operation, the most important property of the detectors to be used in the experimental setup is a combination of the short response of the detectors and their high time resolution. The radiation tolerance to a harsh irradiation environment remains mandatory but not the main factor of the collider’s experiments using electronic beams. A short response in combination with high time resolution ensures minimization of the influence of the pile-up and spill-over effects at the high frequency of collisions (higher than 50 MGz). The radiation hardness of the materials maintains the long-term high accuracy of the detector calibration. This paper discusses the prospects for using modern inorganic scintillation materials for calorimetric detectors at future colliders.

Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes

Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (Vm) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FASTM). Different from present multiplexing approaches, FASTM uses phase-sensitive signal detection, which renders various combinations of common probes for Vm and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FASTM allows simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity and minimal crosstalk. FASTM is also suited for multiplexing using single-cell microscopy and genetically-encoded FRET biosensors. Moreover, FASTM is compatible with opto-chemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FASTM also allows resolving rapid chemical reactions. Altogether, FASTM opens new opportunities for interrogating cellular signaling.