Measurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy.

ABSTRACT: A new approach is presented for measuring the three-dimensional orientation of individual macromolecules using single molecule fluorescence polarization (SMFP) microscopy. The technique uses the unique polarizations of evanescent waves generated by total internal reflection to excite the dipole moment of individual fluorophores. To evaluate the new SMFP technique, single molecule orientation measurements from sparsely labeled F-actin are compared to ensemble-averaged orientation data from similarly prepared densely labeled F-actin. Standard deviations of the SMFP measurements taken at 40 ms time intervals indicate that the uncertainty for individual measurements of axial and azimuthal angles is approximately 10 degrees at 40 ms time resolution. Comparison with ensemble data shows there are

SUBMITTER: Forkey JN 

PROVIDER: S-EPMC1366610 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature