Improved Super-Resolution Imaging in Heavy Water

Abstract

Advanced fluorescence microscopy techniques including single-molecule and super-resolution imaging require bright and photostable dyes that can be selectively targeted to biomolecules. There is therefore an ongoing interest in the development of improved chromophores for biology, especially ones that absorb and emit in the near-infrared. Single fluorophore brightness is a key parameter in localization-based super-resolution imaging techniques such as (F)PALM, (d)STORM, or GSDIM: the brighter the emitter, the more precisely it can be localized and the better the resolution is in the final image. Nonetheless, with few exceptions, most commercially available fluorophores excitable beyond 630 nm have poor fluorescence quantum yields with values around 0.3 or lower limiting their brightness.We introduce a simple, cost-effective, and biocompatible method to enhance the fluorescence quantum yield of several commercially available oxazine (ATTO655, ATTO680, ATTO700) and cyanine (Cy5, Alexa Fluor 647, Cy5.5, Alexa Fluor 700, Cy7) fluorophores [1, 2]. We demonstrate that for all oxazines and one cyanine, the fluorescence quantum yield more than doubles in heavy water (D2O) compared to water (H2O). This increase leads to the detection of twice as many photons per molecule and to a corresponding improvement in the mean localization precision, which enables higher resolution in biological subdiffraction imaging. We demonstrate that super-resolution imaging in D2O can be used to resolve diffraction-limited subcellular structures such as filopodia or microtubules and by performing quantitative morphological analysis of ligand-induced clustering of arrestin proteins upon G protein-coupled receptor stimulation [3].[1] S. F. Lee, Q. Verolet, A. Furstenberg, Angew. Chem. Int. Ed. 2013, 52, 8948.[2] K. Klehs, C. Spahn, U. Endesfelder, S. F. Lee, A. Furstenberg, M. Heilemann, submitted.[3] Z. Truan, L. Tarancon Diez, C. Bonsch, S. Malkusch, U. Endesfelder, M. Munteanu, O. Hartley, M. Heilemann, A. Furstenberg, J. Struct. Biol., in press.