Jihee Hwang
Jinho Park
Keewon Sung
So Young Bak
The benefit of single molecule measurement is observing the motion of “individual” molecules whereas bulk (ensemble) measurements only allow viewing of “average” properties. Through single molecule methods, it is possible to directly obtain information about heterogeneity of each molecule in ensemble, to track “true” time trajectories of single molecule, to detect multiple kinetic paths by transient intermediate states in reaction mechanisms and more. Among a variety of single molecule methods, TIRF (Total Internal Reflection Fluorescence) is a novel technique to study various dynamics of single molecules by observing the special thin part of cell-substrate region through emission of fluorophores attached on the surface. Since TIRF uses evanescent wave, which is formed when light is internally reflected off of an interface between specimen and glass(or qurtz) at an angle greater than the critical angle of total internal reflection, near the surface?within 100nm?it is possible to detect the events of thin cell surface through selective excitation of fluorophores in aqueous and cellular environments.
We study TIRF integrated with FRET (Fluorescence Resonance Energy Transfer) which is defined as a function of distance and energy transferred between a donor chromophore and an acceptor chromophore (in close proximity, typically <10nm) through nonradiative dipole-dipole coupling. In our experiments, we observe the real-time DNA replication by applying TIRF to a surface immobilized sample. DNA replication is the essential process of transferring genetic information of living organisms by copying DNA with the help of DNA polymerase. Among the factors relevant in this process, we can specifically investigate the dynamic motion of DNA polymerase. We demonstrate the effect of Mg2+ and dNTP on the replication rate through real-time monitoring of the DNA copying process which provides a glimpse of these complex dynamical motions involving DNA polymerase.
Single Molecule Spectroscopy
Super-resolution Optical & Nano-optical Nanoscopy
Real-time Femtosecond Dynamics