Single Molecule Dynamics Studied by Polarization Modulation

Abstract

We observed and made unambiguous distinctions between abrupt photophysical events of single molecules: a rotational jump of a single dipole, a transition to a dark state (reversible and irreversible photobleaching), and a spectral jump. The study was performed in the far field by modulating the excitation polarization and monitoring the fluorescence in time. This technique also allowed us to measure the in-plane dipole orientation of stationary single molecular dipoles with subdegree accuracy and to resolve desorption and readsorption of fluorophores from and onto a glass surface. In one case, clear evidence was obtained for rapid rotation of the dipole after a desorption process.

Optical Studies of Single Molecules at Room Temperature

“…Figure 5 shows a sequence of images of a blinking single GFP. Similar but less frequent blinking behavior has also been seen for some but not all single choromophores (42). A general strategy to distinguish photoinduced and spontaneous processes is to check the excitation intensity dependence.…”

“…Similarly, the emission intensities of single molecules fluctuate as the result of the spectral changes (17,42). These intrinsic fluctuations contain rich dynamic information of single molecules and their interacting environments.…”

“…By using a polarization modulation scheme, Ha et al directly observed rotational jumps of single fluorophores on a surface (42). Recently, they developed a more sophisticated scheme to monitor free and hindered rotation (64).…”

Simultaneous, accurate measurement of the 3D position and orientation of single molecules

“…However, immobile fluorescing SMs produce an inherently anisotropic emission pattern that depends on the orientation of the SM emission dipole moment relative to the optical axis (7,8). The work by Enderlein et al (9) has shown that fitting such an SM image to a 2D Gaussian can result in position errors of tens of nanometers for molecules located in the microscope's focal plane.…”

Stepping rotation of F 1 -ATPase visualized through angle-resolved single-fluorophore imaging

“…Individual behaviors can be assessed by single-fluorophore imaging (1)(2)(3)(4)(5), which is much less perturbing than imaging through a huge tag such as a plastic bead or actin filament (6). Real-time determination of fluorophore orientation (5,7,8) should be particularly useful, because a conformational change necessarily accompanies reorientation of one part against others. Thus far, however, successful applications of this potentially powerful method have been made infrequently.…”