Tethered particle microscopy is a powerful tool to study the dynamics of DNA molecules and DNA-protein complexes in single-molecule experiments. We demonstrate that stroboscopic total internal reflection microscopy can be used to characterize the three-dimensional spatiotemporal motion of DNA-tethered particles. By calculating characteristic measures such as symmetry and time constants of the motion, well-formed tethers can be distinguished from defective ones for which the motion is dominated by aberrant surface effects. This improves the reliability of measurements on tether dynamics. For instance, in observations of protein-mediated DNA looping, loop formation is distinguished from adsorption and other nonspecific events.

We have applied tethered particle microscopy (TPM) as a single molecule analysis tool to studies of the conformational dynamics of poly-uridine(U) messenger (m)RNA and 16S ribosomal (r)RNA molecules. Using stroboscopic total internal reflection illumination and rigorous selection criteria to distinguish from nonspecific tethering, we have tracked the nanometer-scale Brownian motion of RNA-tethered fluorescent microspheres in all three dimensions at pH 7.5, 22 degrees C, in 10 mM or 100 mM NaCl in the absence or presence of 10 mM MgCl(2). The addition of Mg(2+) to low-ionic strength buffer results in significant compaction and stiffening of poly(U) mRNA, but not of 16S rRNA. Furthermore, the motion of poly(U)-tethered microspheres is more heterogeneous than that of 16S rRNA-tethered microspheres. Analysis of in-plane bead motion suggests that poly(U) RNA, but less so 16S rRNA, can be modeled both in the presence and absence of Mg(2+) by a statistical Gaussian polymer model. We attribute these differences to the Mg(2+)-induced compaction of the relatively weakly structured and structurally disperse poly(U) mRNA, in contrast to Mg(2+)-induced reinforcement of existing secondary and tertiary structure contacts in the highly structured 16S rRNA. Both effects are nonspecific, however, as they are dampened in the presence of higher concentrations of monovalent cations.

We present a total internal reflection fluorescence microscopy (TIRFM) technique for quantitative molecular-level investigations of macromolecular adsorption and interactions at liquid-liquid interfaces.The technique provides the ability to selectively excite species within tens of nanometers of the interface and is an excellent tool for nonintrusive in situ investigations. The apparatus uses an oil-water assembly that is approximately 1.0 mm thick, enabling us to achieve a diffusion time constant of e3 min, an equilibrium adsorption time on the order of minutes rather than hours, and the possibility of using hydrodynamic shear to create new interfaces. In this paper, we give a detailed description of the apparatus and present some preliminary results of a study on the equilibrium adsorption of bovine serum albumin (BSA) and lysozyme at the oil-water interface.

We present experimental results of the effect of fluorescent labels on the adsorption kinetics and diffusion of bovine serum albumin (BSA) at the oil−water interface. We performed comparative studies on BSA labeled with exactly 1, an average of 1.7, and exactly 2 fluorescein-5-isothiocyanate (FITC) molecules. We used total internal reflection fluorescence microscopy along with fluorescence photobleaching recovery as an in-situ, noninvasive measure of diffusion and adsorption of proteins at the interface. We used ion-exchange chromatography to exploit the difference in electronegativity of proteins with different labeling ratios to effect the separation required to prepare the monodisperse (single- and double-labeled) samples. Absorbance spectroscopy measurements at 278 nm (BSA) and 490 nm (FITC) were used to calibrate the eluant from the chromatography column and determine the labeling ratio. The results showed that the attachment of an extrinsic label has a pronounced effect on both adsorption and diffusion of proteins. For instance, the apparent diffusion coefficient of a BSA molecule conjugated with 2 FITC molecules was estimated to be 40% greater than that of BSA, to which only a single label had been attached. The effects of concentration quenching on the fluorescence recovery after photobleaching were examined, and the recovery curves were shown to be free of quenching effects, even at a labeling ratio of 2.

Optical tweezers are a powerful tool for the study of single biomolecules. Many applications require that a molecule be held under constant tension while its extension is measured. We present two schemes based on scanning-line optical tweezers to accomplish this, providing all-optical alternatives to force-clamp traps that rely on electronic feedback to maintain constant-force conditions for the molecule. In these schemes, a laser beam is rapidly scanned along a line in the focal plane of the microscope objective, effectively creating an extended one-dimensional optical potential over distances of up to 8 microm. A position-independent lateral force acting on a trapped particle is created by either modulating the laser beam intensity during the scan or by using an asymmetric beam profile in the back focal plane of the microscope objective. With these techniques, forces of up to 2.69 pN have been applied over distances of up to 3.4 microm with residual spring constants of <26.6 fN/microm. We used these techniques in conjunction with a fast position measurement scheme to study the relaxation of lambda-DNA molecules against a constant external force with submillisecond time resolution. We compare the results to predictions from the wormlike chain model.