Intramolecular fluorescence correlation spectroscopy in a feedback tracking microscope.
We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that in comparison to traditional fluorescence correlation spectroscopy, tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity, and long-timescale decays. We demonstrate our approach using a three-dimensional tracking microscope to study genomic lambda-phage DNA molecules with various fluorescence label configurations.
McHale
Kevin
K
Edward L. Ginzton Laboratory, Stanford University, Stanford, California, USA. mchalek@gmail.com
Mabuchi
Hideo
H
D010582:Bacteriophage lambda; D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D016014:Linear Models; D008853:Microscopy / Q000379:methods*; D008968:Molecular Conformation; D009068:Movement; D017785:Photons; D013050:Spectrometry, Fluorescence / Q000379:methods*
D004279:DNA, Viral; D005456:Fluorescent Dyes