IPALM: 3D Optical Imaging of Protein Locations at the Nanometer Level

Abstract

Accurate determination of protein locations in 3D gives insight to cellular organization on the molecular scale. Here we describe a single photon, simultaneous multi-phase interferometric technique, providing 10-nm vertical localization. When combined with photoactivated localization microscopy PALM which can provide molecular coordinate based 2D resolution, a new technique termed iPALM resolves 3D molecular coordinates of individual fluorescent protein-tagged proteins with sub-20 nm resolution. The excellent photon sensitivity enables it to maintain these high resolution standards with the less bright but biologically preferable endogeously labeled fluorescent proteins. This technique is applied to plasma membranes, microtubules, endoplasmic reticulum, and focal adhesions. In the focal adhesions several protein specific layers can be characterized with < 4 nm reproducibiliy using iPALM.