PT  - JOURNAL ARTICLE
AU  - Shama Sograte-Idrissi
AU  - Nazar Oleksiievets
AU  - Sebastian Isbaner
AU  - Mariana Eggert-Martinez
AU  - Jörg Enderlein
AU  - Roman Tsukanov
AU  - Felipe Opazo
TI  - Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors
AID  - 10.1101/500298
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 500298
4099  - http://biorxiv.org/content/early/2018/12/19/500298.short
4100  - http://biorxiv.org/content/early/2018/12/19/500298.full
AB  - DNA-PAINT is a rapidly developing fluorescence super-resolution technique which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using 3 nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the 3 targets with 20 nm resolution, and within only 35 minutes acquisition time.