Defocused Imaging in Wide-field Fluorescence Microscopy

Abstract

Defocused imaging in wide-field fluorescence microscopy provides information on the 3D orientation of the transition dipole moment of single molecules with nanometer spatial resolution. In this chapter, the theoretical background of defocused imaging will be presented, followed by two experimental applications. As a first example, defocused imaging was used along with fluorescence lifetime measurements to prove the fact that the molecular orientation of dye molecules in a thin film has a significant affect on their fluorescence lifetime. This is attributed to the electromagnetic boundary condition effect. As a second example, the power of the technique for following 3D molecular rotational reorientation (molecular rotational diffusion) in thin polymer films is demonstrated. Since many molecules can be monitored in parallel, both the temporal and the spatial heterogeneity of polymer dynamics can be addressed.