Single-molecule techniques in biophysics: a review of the progress in methods and applications (1704.06837v2)

Abstract: Single-molecule biophysics has transformed our understanding of the fundamental molecular processes involved in living biological systems, but also of the fascinating physics of life. Far more exotic than a collection of exemplars of soft matter behaviour, active biological matter lives far from thermal equilibrium, and typically covers multiple length scales from the nanometre level of single molecules up several orders of magnitude to longer length scales in emergent structures of cells, tissues and organisms. Biological molecules are often characterized by an underlying instability, in that multiple metastable free energy states exist which are separated by energy levels of typically just a few multiples of the thermal energy scale of kBT, where kB is the Boltzmann constant and T the absolute temperature, implying complex, dynamic inter-conversion kinetics across this bumpy free energy landscape in the relatively hot, wet environment of real, living biological matter. The key utility of single-molecule biophysics lies in its ability to probe the underlying heterogeneity of free energy states across a population of molecules, which in general is too challenging for conventional ensemble level approaches which measure mean average properties. Parallel developments in both experimental and theoretical techniques have been key to the latest insights and are enabling the development of highly-multiplexed, correlative techniques to tackle previously intractable biological problems. Experimentally, technological developments in the sensitivity and speed of biomolecular detectors, the stability and efficiency of light sources, probes and microfluidics, have enabled and driven the study of heterogeneous behaviours both in vitro and in vivo that were previously undetectable by ensemble methods...

Insights into Single-Molecule Techniques in Biophysics and Their Applications

The reviewed article provides a comprehensive examination of single-molecule techniques that have fundamentally altered our understanding of biophysics by enabling the observation of molecular behavior and interactions at an unprecedented level of detail. Single-molecule techniques have emerged as a transformative approach in biophysics by allowing researchers to observe heterogeneity in biological systems that are often obscured when using traditional ensemble methods. This review systematically discusses the experimental and computational developments that have expanded the scope and resolution of single-molecule studies, covering both the technical advances and the biological questions they address.

Key Advances in Experimental Techniques

Single-molecule detection techniques, such as super-resolution microscopy, have significantly enhanced the ability to visualize molecules in vitro and in vivo. One of the primary focuses of this review is the improvement in light microscopy approaches, including techniques like photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). These techniques leverage the photochemical properties of fluorophores to achieve optical resolutions well beyond the diffraction limit, thereby enabling the visualization of individual biomolecules within their native environments. Another important area of advancement is the development of tools that manipulate molecules, such as force spectroscopy techniques. Optical and magnetic tweezers, as well as atomic force microscopy (AFM), are pivotal in measuring forces and torques on biomolecules and have notably advanced our understanding of molecular motors and protein folding mechanisms.

The review also highlights innovations in hybrid and correlative techniques, where multiple methodologies are combined to provide complementary datasets. For instance, integrating AFM with fluorescence microscopy allows concurrent visualization of molecular dynamics alongside mechanically induced conformations. Similarly, patch clamp techniques combined with single-molecule imaging enable the paper of ion channel behaviors at single-molecule resolution, allowing insights into how conformational changes relate to ion flow.

Computational Advances and Simulation Techniques

The fusion of experiment and computation is a cornerstone of current biophysical research, providing a means to cross-validate experimental data and extend insights into molecular dynamics beyond experimental limits. Advances in computational techniques, notably molecular dynamics (MD) simulations, have been instrumental in exploring time-dependent biological processes. Developments in coarse-grained simulations, such as the oxDNA model, facilitate the paper of large biomolecular complexes while maintaining a reasonable level of detail. These simulations are crucial for understanding processes that occur over longer time scales or involve large systems that are otherwise computationally prohibitive to model at an atomistic level.

Quantum mechanical simulations, though computationally demanding, are gaining traction in elucidating the intricate details of enzyme catalysis and electron transport processes. The application of path integral molecular dynamics (PIMD) has provided novel insights, particularly regarding the quantum behavior of protons in enzyme active sites, which traditional classical simulations might overlook.

Practical and Theoretical Implications

The transition from single-molecule proof-of-principle studies to applications that address complex biological questions signifies the maturation of these techniques. As single-molecule studies are inherently low throughput, there is a push towards multiplexing methods to achieve statistically significant data. This includes high-throughput adaptations of existing techniques and innovations such as microfluidics for parallel analysis and efficiency enhancements.

Moreover, single-molecule biophysics has practical implications for biotechnology and medicine, particularly in areas such as personalized medicine and targeted therapies. Advances in DNA sequencing, biosensing, and diagnostic techniques rely heavily on the ability to paper interactions at the single-molecule level, promising a more precise understanding and utilization of molecular diagnostics in clinical settings.

Future Directions and Challenges

Future challenges in single-molecule biophysics include managing and analyzing large datasets, ensuring reproducibility, and adapting complex experimental setups for broader use within the scientific community. The integration of AI and machine learning algorithms offers potential in automating data analysis and feature extraction, enhancing both the accuracy and throughput of single-molecule studies.

In conclusion, the reviewed article underscores the pivotal role of single-molecule techniques in current and future biophysical research. By bridging gaps between molecular biology, physics, and computational science, these techniques continue to provide deep insights into the fundamental processes of life, pushing the boundaries of what can be observed and understood at the molecular level.