Optical manipulation and imaging of
single biological molecules

Marco Capitanio

Mo, 7 Mar 2016, at 10:00. Aula Querzoli

pdf of the presentation

Single molecule (SM) techniques have greatly developed over the past twenty years to respond to the need of overcoming some of the
limitations of traditional, bulk solution measurements. The manipulation of single biological molecules has created the opportunity to
measure mechanical properties of biopolymers and control the mechanical parameters of protein-protein and protein-DNA interactions.
SM fluorescence detection, on the other hand, represents an incredibly versatile tool for studying protein activity in vitro and in vivo, leading to the possibility of localizing and tracking single molecules with nanometer precision.

In this lecture I will introduce optical techniques for the manipulation and imaging of single biological molecules. I will describe physical principles at the base of trapping by optical tweezers, capabilities and limitations in terms of spatial and temporal resolution, and the application of optical tweezers to the study of molecular motors (proteins responsible for cell locomotion and intra-cellular trafficking) and DNA-binding proteins involved in gene expression regulation. I will then illustrate techniques for the imaging and localization of single biological molecules using fluorescence microscopy. Through fitting of the instrument point-spread-function to
the SM image, in fact, one can accomplish localization with a precision depending mainly on signal-to-noise ratio (SNR) and reaching a limit
of about one nanometer. These methodologies find powerful applications in the study of the dynamics of motor proteins, as well as of the diffusion processes underlying target search in DNA-binding proteins. Moreover, when combined with the use of photo-switchable or photo-activable chromophores, nanometer localization of single chromophores allowed the development of super-resolution microscopy techniques such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM).

M. Capitanio and F. S. Pavone, "Interrogating biology with force: single-molecule high-resolution measurements with optical tweezers", Biophys. J. 105, 1293 (2013)

G. Patterson et al., "Superresolution Imaging using Single-Molecule Localization", Annu. Rev. Phys. Chem. 61, 345 (2010)