BREAKING THE RESOLUTION BARRIER IN OPTICAL MICROSCOPY: A NEW RESOLUTION MEASURE WITH APPLICATIONS TO SINGLE MOLECULE IMAGING

Rayleigh´s resolution criterion, although extensively used in optical microscopy, is well known to be based on heuristic notions. In fact, recent single molecule experiments have shown that this criterion can be surpassed in a regular optical microscope. The inadequacy of Rayleigh´s criterion has necessitated a reassessment of the resolution limits of optical microscopes. Recently, we proposed a new resolution measure that overcomes the limitations of Rayleigh´s criterion. Known as the fundamental resolution measure FREM, our new result predicts that distances well below Rayleigh´s limit can be resolved in an optical microscope. The effect of deteriorating experimental factors on the new resolution measure is also investigated. Further, it is experimentally verified that distances well below Rayleigh´s limit can be measured from images of closely spaced single molecules with an accuracy as predicted by the new resolution measure. We have also addressed an important problem in single molecule microscopy that concerns the accuracy with which the location of a single molecule can be determined. In particular, we have derived analytical expressions for the limit to the 2D/3D localization accuracy of a single molecule.