Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

It has been over 20 years since the pioneering work of Arthur Ashkin, and in the intervening years, the field of optical tweezers has grown tremendously. Optical tweezers are now being used in the investigation of an increasing number of biochemical and biophysical processes, from the basic mechanical properties of biological polymers to the multitude of molecular machines that drive the internal dynamics of the cell. Innovation, however, continues in all areas of instrumentation and technique, with much of this work focusing on the refinement of established methods and on the integration of this tool with other forms of single-molecule manipulation or detection. Although technical in nature, these developments have important implications for the expanded use of optical tweezers in biochemical research and thus should be of general interest. In this review, we address these recent advances and speculate on possible future developments.

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Recent Advances in Optical Tweezers

We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, whereas maximum-likelihood fitting is practically optimal.

Optimized localization analysis for single-molecule tracking and super-resolution microscopy.

Optical tweezers have become one of the primary weapons in the arsenal of biophysicists, and have revolutionized the new field of single-molecule biophysics. Today's techniques allow high-resolution experiments on biological macromolecules that were mere pipe dreams only a decade ago.

Optical tweezers study life under tension

Single-molecule methods have matured into powerful and popular tools to probe the complex behaviour of biological molecules, due to their unique abilities to probe molecular structure, dynamics and function, unhindered by the averaging inherent in ensemble experiments. This review presents an overview of the burgeoning field of single-molecule biophysics, discussing key highlights and selected examples from its genesis to our projections for its future. Following brief introductions to a few popular single-molecule fluorescence and manipulation methods, we discuss novel insights gained from single-molecule studies in key biological areas ranging from biological folding to experiments performed in vivo.

https://royalsocietypublishing.org/doi/pdf/10.1098/rsif.2007.1021

Single-molecule biophysics: at the interface of biology, physics and chemistry

Mechanical drift is a long-standing problem in optical microscopy that occurs in all three dimensions. This drift increasingly limits the resolution of advanced surface-coupled, single-molecule experiments. We overcame this drift and achieved atomic-scale stabilization (0.1 nm) of an optical microscope in 3D. This was accomplished by measuring the position of a fiducial mark coupled to the microscope cover slip using back-focal-plane (BFP) detection and correcting for the drift using a piezoelectric stage. Several significant factors contributed to this experimental realization, including (i) dramatically reducing the low frequency noise in BFP detection, (ii) increasing the sensitivity of BFP detection to vertical motion, and (iii) fabricating a regular array of nanometer-sized fiducial marks that were firmly coupled to the cover slip. With these improvements, we achieved short-term (1 s) stabilities of 0.11, 0.10, and 0.09 nm (rms) and long-term (100 s) stabilities of 0.17, 0.12, and 0.35 nm (rms) in x, y, and z, respectively, as measured by an independent detection laser.

http://web.missouri.edu/~kinggm/Carter_2007b.pdf

David Alchenberger

Papers

Stabilization of an optical microscope to 0.1 nm in three dimensions