© 2012 Nature America, Inc. All rights reserved. BRIEF COMMUNICATIONS NATURE METHODS | ADVANCE ONLINE PUBLICATION | 1 isoforms. Nanobody-mediated targeting of organic dyes to GFP- fusion constructs would combine the molecular specificity of genetic tagging with the high photon yield of organic dyes and minimal linkage error. We covalently coupled anti-GFP nanobodies to Alexa Fluor 647 (AF647) and added them to MDCK cells expressing GFP anchored via glycosylphosphatidylinositol to the plasma mem- brane (GPI-GFP). The nanobodies bound to transfected cells readily at low concentrations but did not bind to untransfected cells (Supplementary Fig. 1). Labeled nanobodies exhibited highly specific and saturable binding at about 0.6 nanobodies per GFP (Online Methods). Under specific buffer conditions, many organic dyes become photoswitchable 6,7 . This fact is the basis for several single-molecule nanoscopy approaches 2–4 . To compare the nanobody-based approach to established methods, we labeled microtubules in fixed Ptk2 cells that stably expressed tubulin-YFP with AF647–anti-GFP nanobod- ies and imaged these cells by single-molecule nanoscopy. Individual microtubules were densely labeled with a full-width half maximum (FWHM) of 26.9 nm 3.7 nm (s.d.) (Fig. 1a), compatible with a microtubule diameter of 25 nm. This was significantly less (Fig. 1a) than what we achieved in parallel experiments using AF647-coupled secondary antibodies to detect microtubules either via anti-GFP (42.7 nm 7.0 nm) or anti-tubulin (45.6 nm 5.8 nm) antibodies, suggesting that the use of nanobodies led to minimal linkage error. We next used the bright AF647–anti-GFP nanobodies for three-dimensional single-molecule nanoscopy with the bi-plane method 8 , which requires thousands of detected photons for good axial resolution. Nanobody-mediated labeling of microtubules in Ptk2 cells allowed us to resolve crossing microtubules with an axial separation of ~100 nm (Fig. 1b). The use of bright labels not only increased localization preci- sion (statistics in Supplementary Fig. 2), but also allowed the high frame rates required for imaging dynamic (live) samples. To realize this advantage, we transiently expressed GPI-GFP in primary hippocampal rat neurons and detected it via AF647– anti-GFP nanobodies. Single-particle tracking PALM (sptPALM) 9 allowed us to follow the trajectories of hundreds of molecules in the neuronal plasma membrane. We assembled a time series of super-resolution images and detected dynamic changes in the neuronal morphology over time (Fig. 1c). Next, we used nanobodies for dual-color imaging. We realized this approach by using anti-GFP nanobodies labeled with Alexa Fluor 700 (AF700) and anti-RFP nanobodies (that also recognize monomeric (m)Cherry) labeled with AF647. We then labeled CV-1 cells expressing Septin7-mCherry and Caveolin1-YFP with these labels and could image both structures with high resolution and specificity (Fig. 1d). A simple, versatile method for GFP-based super-resolution microscopy via nanobodies Jonas Ries, Charlotte Kaplan, Evgenia Platonova, Hadi Eghlidi & Helge Ewers We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super- resolution imaging. The single-molecule localization–based super-resolution imag- ing techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy 1–4 (here referred to as ‘single-molecule nanoscopy’) require specific and efficient delivery of bright fluorophores into close proximity of the target structure without adding substantial background. Present labeling techniques usually involve a tradeoff between these properties. Bright organic dyes can be delivered via anti- bodies, but their large size displaces the dye from the target in a ‘linkage error’ of ~10 nm, and highly specific antibodies are avail- able only for some proteins. Methods based on photoactivatable fluorescent proteins or enzymatic labeling schemes demand the generation of new fusion constructs, which should be character- ized and may not be functional. We demonstrate a simple, efficient and versatile method that optimizes several labeling parameters and can be used with any of the thousands of functionally tested GFP constructs available. We used very small and high-affinity camelid antibodies (nanobod- ies) to GFP 5 to deliver bright organic fluorophores to GFP-tagged proteins for use in single-molecule nanoscopy. Organic dyes are conventionally delivered to target struc- tures via antibodies, which are ~150 kDa, ~10 nm in size and of varying affinity. In contrast, the 13-kDa anti-GFP nanobod- ies are smaller (1.5 nm × 2.5 nm) and have a high affinity for GFP (0.59 nM; ref. 5; Supplementary Fig. 1) and several of its Institute of Biochemistry and Laboratory of Physical Chemistry, ETH Zurich, Zurich, Switzerland. Correspondence should be addressed to H.Ewers (helge.ewers@bc.biol.ethz.ch). RECEIVED 2 DECEMBER 2011; ACCEPTED 28 MARCH 2012; PUBLISHED ONLINE 29 APRIL 2012; DOI:10.1038/NMETH.1991