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

The advent of single molecule optics has had a profound impact in fields ranging from biophysics to material science, photophysics, and quantum optics. However, all existing room-temperature single molecule methods have been based on fluorescence detection of highly efficient emitters. Here we demonstrate that standard, modulation-free measurements known from conventional absorption spectrometers can indeed detect single molecules. We report on quantitative measurements of th...

Single molecule fluorescence tracking provides information at nm-scale and ms-temporal resolution about the dynamics and interaction of individual molecules in a biological environment. While the dynamic behavior of isolated molecules can be characterized well, the quantitative insight is more limited when interactions between two indistinguishable molecules occur. We address here this aspect by developing a solid theoretical foundation for a spectroscopy of interaction times...

One of the most intriguing results of single molecule experiments on proteins and nucleic acids is the discovery of functional heterogeneity: the observation that complex cellular machines exhibit multiple, biologically active conformations. The structural differences between these conformations may be subtle, but each distinct state can be remarkably long-lived, with random interconversions between states occurring only at macroscopic timescales, fractions of a second or lon...

We describe a simple automated method to extract and quantify transient heterogeneous dynamical changes from large datasets generated in single molecule/particle tracking experiments. Based on wavelet transform, the method transforms raw data to locally match dynamics of interest. This is accomplished using statistically adaptive universal thresholding, whose advantage is to avoid a single arbitrary threshold that might conceal individual variability across populations. How t...

Although significant insight has been gained into biochemical, genetic and structural features of oxidative phosphorylation (OXPHOS) at the single-enzyme level, relatively little was known of how the component complexes function together in time and space until recently. Several pioneering single-molecule studies have emerged over the last decade in particular, which have illuminated our knowledge of OXPHOS, most especially on model bacterial systems. Here, we discuss these r...

In the past one hundred years, deterministic rate equations have been successfully used to infer enzyme-catalysed reaction mechanisms and to estimate rate constants from reaction kinetics experiments conducted in vitro. In recent years, sophisticated experimental techniques have been developed that allow the measurement of enzyme- catalysed and other biopolymer-mediated reactions inside single cells at the single molecule level. Time course data obtained by these methods are ...

Fluctuation in fluorescence emission of immobilized single molecule is typically ascribed to the chromophore's intrinsic structural conformations and the influence of local environmental factors. Despite extensive research over several decades since its initial observation, a direct connection between these spectral fluctuations and the rearrangement of emission dipole orientations has remained elusive. In this study, we elucidate this fundamental molecular behavior and its u...

Over the past decade, advances in super-resolution microscopy and particle-based modeling have driven an intense interest in investigating spatial heterogeneity at the level of single molecules in cells. Remarkably, it is becoming clear that spatiotemporal correlations between just a few molecules can have profound effects on the signaling behavior of the entire cell. While such correlations are often explicitly imposed by molecular structures such as rafts, clusters, or scaf...

To illustrate the power of the biophysical approach in solving important problems in life science, I present here one of our current research projects as an example. We have developed special biophotonic techniques to study the dynamic properties of signaling proteins in a single living cell. Such a study allowed us to gain new insight into the signaling mechanism that regulates programmed cell death.

Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the...