Although Single Molecule Localization (SML) techniques have pushed the resolution of fluorescence microscopy beyond the diffraction limit, the accuracy of SML has been limited by the brightness of the fluorophores. The introduction of Quantum Dots (QD) for SML promises to overcome this barrier, and the QD Blueing technique provides a novel approach to SML microscopy. QDs have a higher quantum yield and absorption cross-section, making them brighter, thereby providing a higher accuracy of localization. The blueing technique is also faster and more quantitative than other SML techniques such as dSTORM. The initial bleaching step required by dSTORM is not necessary and each QD is imaged only once as its emission spectrum moves through the blueing window in contrast to dSTORM where the same molecule might be imaged multiple times. Single color QD Blueing has been demonstrated. However in biological imaging, multi-color imaging is essential for understanding the samples under study. Here we introduce two color superresolution microscopy using QD Blueing on biological samples. We demonstrate simultaneous imaging of microtubules and mitochondria in HepG2 cells with a localization accuracy of 40nm. We further show how QD Blueing can be optimized through the control of the sample mounting medium.

Date Published: 9 March 2015
PDF: 9 pages
Proc. SPIE 9331, Single Molecule Spectroscopy and Superresolution Imaging VIII, 93310C (9 March 2015); doi: 10.1117/12.2077293

Published in SPIE Proceedings Vol. 9331:
Single Molecule Spectroscopy and Superresolution Imaging VIII
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)