Chapter 12: Nanoscale biological fluorescence imaging: breaking the diffraction barrier

Abstract

Biological imaging has been limited by the finite resolution of light microscopy. Recent developments in ultra-high-resolution microscopy methods, many of which are based on fluorescence, are breaking the diffraction barrier; it is becoming possible to image intracellular protein distributions with resolution of tens of nanometers or better. Fluorescence photoactivation localization microscopy (FPALM) is an example of such an ultra-high-resolution method which can image living or fixed cells with demonstrated lateral resolution of better than 20 nm. A detailed description of the methods involved in FPALM imaging of biological samples is presented here, accompanied by comparison with existing methods from the literature.