Combined multiparticle tracking and quasi-elastic light-scattering microscope

Abstract

Fluorescence polarization anisotropy can be used to study collective dynamics over time scales limited by the fluorescence lifetime which is rarely larger than microseconds. Recently, single particle tracking techniques have been developed that follow the motion of a single probe particle with nanometer precision and times as short as milliseconds. By studying only a single particle, the rich heterogeneity of the cell become inaccessible. There is, therefore, a need for the combined study of both collective and individual nm to micron scale fluctuations over a wide range of time scales. Dynamic, or quasi-elastic light scattering is an excellent tool for tracking on time and length scales that are biologically relevant. Its weakness has been the difficulties of interpreting its data set when the scattering particles are confined to fluctuate less than a wavelength and the difficulty of scattering from known regions within single cells. Working on an optical microscope, the authors have solved the problems of knowing the scattering volume and geometry by simultaneously imaging and scattering from the same particles. Recent interpretation developments enable them to invert their scattering data to obtain particle displacements. Significantly, in this experimental geometry, the authors make video recordings of the real space images of the particles they scatter from. From these recordings, the authors simultaneously track many particles in the field of view. The inhomogeneities of the system-for example, are some particles fixed while others move-can be studied by following the tracks of many particle simultaneously. The relative simplicity of this technique is that it uses commercially available detectors, electronics, and video technology.