Three-dimensional Nano-Localization of Fluroescent Emitters

The use of fluorescence light for far-field imaging at the nanometer scale has drawn much attention worldwide. Example techniques include STED microscopy and localization microscopy (PALM, STORM). Typically, nanoscopy approaches have been focused on the manipulation and detection of the intensity of fluorescence light leading to lateral accuracy of a few nanometers. However, three-dimensional nanometer accuracy, in particular axial accuracy of the same order, remains a challenge. The phase of fluorescence light has shown to be valuable for axial measurements at the nano-scale. We extent the framework of phase microscopy to fluorescence light at the single-molecule level and demonstrate an approach combining super resolution microscopy with fluorescence interferometry for localizing a single nano-object to within several nanometres in all three dimensions. In a first proof of principle we demonstrate three-dimensional nano-localization of quantum dots within a standard deviation for the lateral and axial localization in the order of 6 nm and 4 nm, respectively. As the localization precision of the single nano-object in all three dimensions depends on the number of detected photons, we are exploring ways to increase the number of detected photons. As a result, we expect our approach to pave the way for three-dimensional, molecular-resolution fluorescence imaging.