Red light, green light: probing single molecules using alternating-laser excitation.
Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein-nucleic acid interactions.
Santoso
Yusdi
Y
Department of Physics and Interdisciplinary Research Collaboration in Bionanotechnology, Clarendon Laboratory, University of Oxford, Parks Road, Oxford, UK.
Hwang
Ling Chin
LC
Le Reste
Ludovic
L
Kapanidis
Achillefs N
AN
D003116:Color; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005782:Gels / Q000737:chemistry; D007834:Lasers
D005782:Gels