Single-molecule techniques in biophysics: a review of the progress in methods and applications

A method is presented that, when used in conjunction with single molecule experimental techniques, allows for the extraction of rates and mechanical properties of a biomolecule undergoing transitions between mechanically distinct states. This analysis enables the exploration of systems where the lifetimes of survival are of order of the intrinsic time constant of the experimental apparatus; permitting the study of kinetic events whose transition rates are an order of magnitud...

Recent efforts to survey the numerous softwares available to perform single molecule tracking (SMT) highlighted a significant dependence of the outcomes on the specific method used, and the limitation encountered by most techniques to capture fast movements in a crowded environment. Other approaches to identify the mode and rapidity of motion of fluorescently labeled biomolecules, that do not relay on the localization and linking of the images of isolated single molecules are...

Single molecule FRET (fluorescence resonance energy transfer) is a powerful technique for detecting real-time conformational changes and molecular interactions during biological reactions. In this review, we examine different techniques of extending observation times via immobilization and illustrate how useful biological information can be obtained from single molecule FRET time trajectories with or without absolute distance information.

The search for what differentiates inanimate matter from living things began in antiquity as a search for a "fundamental life force" embedded deep within living things - a special material unit owned only by life - later transforming to more circumspect search for unique gains in function that transform nonliving matter to that which can reproduce, adapt, and survive. Aristotelian thinking about the matter/life distinction and Vitalistic philosophy's "vital force" persisted w...

Recent advances in non-equilibrium statistical mechanics and single molecule technologies make it possible to extract free energy differences from irreversible work measurements in pulling experiments. To date, free energy recovery has been focused on native or equilibrium molecular states, whereas free energy measurements of kinetic states (i.e. finite lifetime states that are generated dynamically and are metastable) have remained unexplored. Kinetic states can play an impo...

There are many processes in biology in which mechanical forces are generated. Force-bearing networks can transduce locally developed mechanical signals very extensively over different parts of the cell or tissues. In this article we conduct an overview of this kind of mechanical transduction, focusing in particular on the multiple layers of complexity displayed by the mechanisms that control and trigger the conversion of a mechanical signal into a biochemical function. Single...

In this paper, single-molecule spectroscopy experiments based on continuous laser excitation are characterized through an open quantum system approach. The evolution of the fluorophore system follows from an effective Hamiltonian microscopic dynamic where its characteristic parameters, i.e., its electric dipole, transition frequency, and Rabi frequency, as well as the quantization of the background electromagnetic field and their mutual interaction, are defined in an extended...

The molecular biology revolution of the last seventy five years has transformed our view of living systems. Scientific explanations of biological phenomena are now synonymous with the identification of the genes, proteins, and signaling molecules involved. The hegemony of the molecular paradigm has only become more pronounced as new technologies allow us to make measurements at scale. Combining this wealth of data with new ``artificial intelligence'' techniques is viewed as t...

Single molecule spectroscopy aims at unveiling often hidden but potentially very important contributions of single entities to a system's ensemble response. Albeit contributing tremendously to our ever growing understanding of molecular processes the fundamental question of temporal evolution, or change, has thus far been inaccessible, resulting in a static picture of a dynamic world. Here, we finally resolve this dilemma by performing the first ultrafast time-resolved transi...

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule F\"orster Resonance Energy Transfer (smFRET) is a powerful approach to inform on the structure and the dynamics of labeled molecules. If the dynamics occur on the sub-millisecond timescale, capturing and quantifying conformational dynamics can be challenging by intensity-based smFRET. Multiparameter fluorescence detection (MFD) addresses this challenge by simultaneously r...