Super-resolution microscopy and single-particle tracking: recent methods we improved with classical statistics
Henrik Flyvbjerg, Department of Micro- and Nanotechnology, Technical University of Denmark
Cosmologists can study the universe because it is transparent and mostly empty. High-energy physics also studies its objects in empty space. The molecular mechanisms of life present a different challenge since they only can be studied in water at physiological temperatures, and they are far too small to be resolved with light microscopes because of Abbe's diffraction limit. This limit is "by-passed" and spatial resolution of a few nanometers is achieved with single-molecule tracking and localization microscopy. This is a hundred times below the diffraction limit. Also, far-field super-resolution techniques now exist, such as photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), which resolves intracellular protein localization patterns to within a few nanometers. In view of their popularity, it is natural to ask whether these techniques are optimal. That is: How does one optimally localize isolated fluorescent beads and molecules imaged as diffraction-limited spots? How does one obtain reliable formulas for the precision of various localization methods? This is a job for physicists, since microscopes and cameras are pure physics and images are digital, pure numbers. I will explain our answers to these questions in terms of basic physics and a little statistics that every experimenter should know. I will close with current research that our answers have precipitated.
1) K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg. Nature Methods 7, 377-381 (2010).
2) News and Views: Nature Methods 7, 357-359 (2010).