Studies of Single Molecules in their Natural Form

Abstract

Single molecule studies make possible the characterization of molecular processes and the identification of biophysical sub‐populations that are not accessible through ensemble studies. We describe tethered particle motion, a method that allows one to study single molecules in their natural form without having to apply any external forces. The method combines darkfield microscopy with a metal nano‐bead. It permits the study of the biophysical properties of the tethered particles, as well as protein–DNA interactions. The method is not suitable for in vivo studies, and we therefore describe two other methods that are appropriate for live‐cell imaging

Dynamic analysis of a diffusing particle in a trapping potential

“…The TPM experimental system details are described elsewhere [25,26]. The particle positions are tracked using a microscope and a CCD camera.…”

Protein-DNA Interactions Studies with Single Tethered Molecule Techniques

“…Such a bead has a significant plasmon scattering which results in an intense signal that can be easily detected by a CCD. (Segall, Nelson et al 2006;Lindner, Nir et al 2009). …”

“…It consists of a dark field (DF) microscope unit (Olympus BX-RLA2, Tokyo, Japan) with a x50 objective lens (NA=0.8) and an EM-CCD camera (Andor DU-885, Belfast, Northern Ireland) with a pixel size of 8x8 m and a maximal pixel read-out rate of 35 MHz (Lindner, Nir et al 2009). …”

“…After incubation of one hour and another wash, the nano beads are introduced. After ~ 30 minutes of incubation and another wash the tethering procedure is complete (Selvin & Ha 2008;Lindner, Nir et al 2009;Zimmermann, Nicolaus et al 2010;Lindner, Nir et al 2011).…”

Plasmonic Scattering as an Efficient Tool for a Force-Free Technique to Follow Single DNA Molecules

“…In solution, the marker moves randomly in a volume that is governed by the restrictions set by the DNA molecule. Tracking and analyzing its position distribution provides an essential tool to follow the dynamics of the DNA conformations [2,3]. Most of the TPM systems use a CCD camera to detect the projected position of the bead on a two-dimensional plane, and the information on the bead height above the surface is lost.…”