Fluorescence Imaging with One Nanometer Accuracy:  Application to Molecular Motors

Abstract

We introduce the technique of FIONA, fluorescence imaging with one nanometer accuracy. This is a fluorescence technique that is able to localize the position of a single dye within ∼1 nm in the x-y plane. It is done simply by taking the point spread function of a single fluorophore excited with wide field illumination and locating the center of the fluorescent spot by a two-dimensional Gaussian fit. We motivate the development of FIONA by unraveling the walking mechanism of the molecular motors myosin V, myosin VI, and kinesin. We find that they all walk in a hand-over-hand fashion.

Single-molecule imaging in live bacteria cells

“…A technique that is being recently employed to many different systems is that of single-molecule localization or 'super-resolution' microscopy, where the high localization accuracy of single-molecule imaging [11] is exploited to build, over time, an image that seems to break the resolution limit. Among these techniques are stochastic reconstruction microscopy (STORM) [12], photoactivated localization microscopy (PALM) [13] and fluorescence PALM [14].…”

Chapter 27 Cell Biology of mRNA Decay

“…As long as discrete signals are sufficiently separated in space (i.e., no overlap of signals within the limits of optical resolution), these can be reordered in a spatially separated manner, and the fitting process will report their position with subwavelength accuracy. For a detailed discussion see Thompson et al (2002) and Yildiz and Selvin (2005). The use of multiple colors (multiplexing) in single molecule experiments promises new quantitative accuracy for colocalization data.…”

“…The use of photoswitchable labels with techniques like PALM and STORM, temporal distances between signals allow construction of high-resolution images of labels that are in closer spatial proximity than the resolution limit (Betzig et al, 2006;Hess et al,2006;Rust et al, 2006). Although some groups use a small number of large pixels (Schmidt et al, 1995), other groups use higher spatial over-sampling (Thompson et al, 2002;Yildiz and Selvin, 2005). To test the experimental procedure, test samples can be prepared with dyes or beads at different concentrations and then imaged in an immobilized state.…”

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

“…Under appropriate conditions, the fluorophores can be located with an accuracy of 1 nm (Churchman et al 2005; Thompson et al 2002;Yildiz and Selvin 2005), thus allowing reliable measurements of inter-dye distances usually in the range of 10-40 nm (i.e., a co-localization technique). When two dyes come into a closer proximity (less than 10 nm) and the donor emission spectra overlaps with the acceptor absorption spectra, a fraction of donorexcited energy may be transferred to an acceptor via Electronic supplementary material The online version of this article (doi:10.1007/s00249-008-0383-z) contains supplementary material, which is available to authorized users.…”

“…In many cases, the information about inter-dye distance is extracted from FRET efficiency histograms which are often approximated by a Gaussian function (Agrawal et al 2008;Coban et al 2006;Deniz et al 1999;Dietrich et al 2002;Ha 2001;Iqbal et al 2008a; Kapanidis et al 2004;Koopmans et al 2007; Kuzmenkina et al 2006;Lee et al 2005;Merchant et al 2007;Murphy et al 2004;Nir et al 2006;Sabanayagam et al 2005;Schuler et al 2005;Sugawa et al 2007;Yildiz and Selvin 2005;Yim et al 2005;Zhang et al 2007). The apparent width of thus extracted histograms is usually in the range of 0.07-0.2 (on a scale of 0-1) and often ascribed to variations of the interdye distance (Coban et al 2006;Dietrich et al 2002;Merchant et al 2007;Schuler et al 2005;Sugawa et al 2007).…”