4 research outputs found
Single-image measurements of monochromatic subdiffraction dimolecular separations
Measuring subdiffraction separations between single fluorescent particles is
important for biological, nano-, and medical-technology studies. Major
challenges include (i) measuring changing molecular separations with high
temporal resolution while (ii) using identical fluorescent labels. Here we
report a method that measures subdiffraction separations between two identical
fluorophores by using a single image of milliseconds exposure time and a
standard single-molecule fluorescent imaging setup. The fluorophores do not
need to be bleached and the separations can be measured down to 40 nm with
nanometer precision. The method is called single-molecule image deconvolution
-- SMID, and in this article it measures the standard deviation (SD) of
Gaussian-approximated combined fluorescent intensity profiles of the two
subdiffraction-separated fluorophores. This study enables measurements of (i)
subdiffraction dimolecular separations using a single image, lifting the
temporal resolution of seconds to milliseconds, while (ii) using identical
fluorophores. The single-image nature of this dimer separation study makes it a
single-image molecular analysis (SIMA) study.Comment: 16 pages, 5 figure
Protein sliding and hopping kinetics on DNA
Using Monte-Carlo simulations, we deconvolved the sliding and hopping
kinetics of GFP-LacI proteins on elongated DNA from their experimentally
observed seconds-long diffusion trajectories. Our simulations suggest the
following results: (1) in each diffusion trajectory, a protein makes on average
hundreds of alternating slides and hops with a mean sliding time of several
tens of ms; (2) sliding dominates the root mean square displacement of fast
diffusion trajectories, whereas hopping dominates slow ones; (3) flow and
variations in salt concentration have limited effects on hopping kinetics,
while in vivo DNA configuration is not expected to influence sliding kinetics;
furthermore, (4) the rate of occurrence for hops longer than 200 nm agrees with
experimental data for EcoRV proteins