Advances in Single-Molecule Fluorescence Methods for Molecular Biology

Abstract

Ever since their introduction two decades ago, single-molecule (SM) fluorescence methods have matured and branched out to address numerous biological questions, which were inaccessible via ensemble measurements. Among the current arsenal, SM fluorescence techniques have capabilities of probing the dynamic interactions of nucleic acids and proteins via Förster (fluorescence) resonance energy transfer (FRET), tracking single particles over microns of distances, and deciphering the rotational motion of multisubunit systems. In this exciting era of transitioning from in vitro to in vivo and in situ conditions, it is anticipated that SM fluorescence methodology will become a common tool of molecular biology.

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

“…3): the extent of the I a 635 increase upon, e.g., the ''parallel'' looping increases from~1.3 to 1.9 with lengthening of the intertarget distance from 247 to 1000 bp. Notably, the increase of I a 635 upon the ''antiparallel'' loop formation is from 8% to 30% smaller than that associated with the ''parallel'' loop-possibly because of the difference of the polarization effects of fluorophore excitation in the twoloop conformations (2,31,32). The expected I a 635 modulation upon looping was estimated by assuming the wormlike chain (WLC) model of DNA flexibility.…”

“…Unless stated otherwise, the center and width of the Gaussian fit were fixed. The single molecule intensity was expressed as (amplitude) Â (width) 2 ). Time trajectories were screened to exhibit characteristic single-molecule FRET features: clear donor-acceptor anticorrelated intensity changes, a typical single-molecule fluorescence intensity, and a single bleaching step.…”

“…In the recent past, fluorescence microscopy of single molecules has become a common mode of experimentation to study molecular interactions and conformational changes, where the unsynchronized behavior of many molecules renders the conventional ensemble measurements insufficient (1,2). Among the different single-molecule (SM) microscopy techniques the Förster resonance energy transfer (FRET) approach is one of the most fruitful (3)(4)(5).…”

Bringing single-molecule spectroscopy to macromolecular protein complexes

“…Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.…”

“…Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] …”