Nanometer-accuracy distance measurements between fluorophores at the single-molecule level
DATE: August 13, 2019
TIME: 9:00am PT, 12:00pm ET, 5:00pm BST
Molecular complexes are major constituents of cells, hence unraveling their mechanisms is key to fuller comprehension of cell biology. Electron Microscopy and X-ray crystallography provide high resolution static images, but deeper understanding requires dynamic measurements.
During the past few decades, we have gained the ability to place small molecules (such as fluorescent probes) on specific sites of molecular machines. However, while single-molecule Förster resonance energy transfer (FRET) can determine relative distance changes between ~2-8 nm, direct distance measurements are difficult to achieve in part because FRET depends on dye orientation and requires careful calibration. Other current super-resolution measurements become error-prone below 25 nm. We developed methods for reliable image registration, and used available information about localization accuracy to measure average distances between fluorophores at sub-nm precision. These procedures can now be used to measure distance between multiple sites of a (protein) complex in different conditions.
This webinar will explain the procedures for sub-nm image registration, explain how distance measurements can be improved by using localization precision information, and show how these procedures can be extended to dynamic distance measurements of biological nano-machines.
Recognition of the potential of "standard" microscopy equipment to deliver nm precision measurements between single fluorophores
Awareness of the impact of local image distortions on registration of multi-channel images of single molecules
Understanding the value of the precision of localization for individual molecules and its impact on distance measurement